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Fluorescence dye-based loop-mediated isothermal amplification (LAMP) is a sensitive nucleic acid detection method, but is limited to single-plex detection and may yield non-specific signals. In this study, we propose a bifunctional probe-based real-time LAMP amplification method for single-plexed or multiplexed detection. The bifunctional probe is derived by modifying the 5′ end of the fluorophore and an internal quencher on one of the LAMP primers; therefore, it can simultaneously be involved in the LAMP process and signal amplification. The fluorescence intensity undergoes a cumulative exponential increase during the incorporation of the bifunctional probe into double-stranded DNA amplicons. The bifunctional probe-based LAMP method is simplified and cost-effective, as the primer design and experimental operations align entirely with the ordinary LAMP. Different from other current probe-based methods, this method does not require additional enzymes, sequences, or special probe structures. Also, it is 10 min faster than several other probe-based LAMP methods. The bifunctional probe-based LAMP method allows the simultaneous detection of the target Vibrio parahaemolyticus DNA and the internal amplification control in a one-pot reaction, demonstrating its potential for multiplexed detection. [ABSTRACT FROM AUTHOR]

Chronic kidney disease (CKD) is one of the most serious non‐communicable diseases affecting the population. In the early‐stages patients have no obvious symptoms, until it becomes life‐threatening leading end‐stage kidney failure. Therefore, it is important to early diagnose CKD to allow therapeutic interventions and progression monitoring. Here, a point‐of‐care (POC) sensing platform is reported for the simultaneous detection of three CKD biomarkers, namely creatinine, urea, and human serum albumin (HSA), using reduced graphene oxide/polydopamine‐molecularly imprinted polymer (rGO/PDA‐MIP) fabricated with novel surface‐molecularly imprinting technology. A multi‐channel electrochemical POC readout system with differential pulse voltammetry (DPV) function is developed, allowing the simultaneous detection of the three biomarkers, in combination with the surface‐MIP electrodes. This sensing platform achieves the record low limit‐of‐detection (LoD) at a femtomolar level for all three analytes, with wide detection ranges covering their physiological concentrations. Clinical validation is performed by measuring these analytes in serum and urine from healthy controls and patients with CKD. The average recovery rate is 81.8–119.1% compared to the results obtained from the hospital, while this platform is more cost‐effective, user‐friendly, and requires less sample‐to‐result time, showing the potential to be deployed in resource‐limited settings for the early diagnosis and tracking progression of CKD. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are promising tools for the early diagnosis of diseases, and bacterial membrane vesicles (MVs) are especially important in health and environment monitoring. However, detecting EVs or bacterial MVs presents significant challenges for the clinical translation of EV-based diagnostics. In this Review, we provide a comprehensive discussion on the basics of nanoplasmonic sensing and emphasize recent developments in nanoplasmonics-based optical sensors to effectively identify EVs or bacterial MVs. We explore various nanoplasmonic sensors tailored for EV or bacterial MV detection, emphasizing the application of localized surface plasmon resonance through gold nanoparticles and their multimers. Additionally, we highlight advanced EV detection techniques based on surface plasmon polaritons using plasmonic thin film and nanopatterned structures. Furthermore, we evaluate the improved detection capability of surface-enhanced Raman spectroscopy in identifying and classifying these vesicles, aided by plasmonic nanostructures. Nanoplasmonic sensing techniques have remarkable precision and sensitivity, making them a potential tool for accurate EV detection in clinical applications, facilitating point-of-care molecular diagnostics. Finally, we summarize the challenges associated with nanoplasmonic EV or bacterial MV sensors and offer insights into potential future directions for this evolving field. [ABSTRACT FROM AUTHOR]

In this study, we report a multiplexed platform for the simultaneous determination of five marine toxins. The proposed biosensor is based on a disposable electrical printed (DEP) microarray composed of eight individually addressable carbon electrodes. The electrodeposition of gold nanoparticles on the carbon surface offers high conductivity and enlarges the electroactive area. The immobilization of thiolated aptamers on the AuNP-decorated carbon electrodes provides a stable, well-orientated and organized binary self-assembled monolayer for sensitive and accurate detection. A simple electrochemical multiplexed aptasensor based on AuNPs was designed to synchronously detect multiple cyanotoxins, namely, microcystin-LR (MC-LR), Cylindrospermopsin (CYL), anatoxin-α, saxitoxin and okadaic acid (OA). The choice of the five toxins was based on their widespread presence and toxicity to aquatic ecosystems and humans. Taking advantage of the conformational change of the aptamers upon target binding, cyanotoxin detection was achieved by monitoring the resulting electron transfer increase by square-wave voltammetry. Under the optimal conditions, the linear range of the proposed aptasensor was estimated to be from 0.018 nM to 200 nM for all the toxins, except for MC-LR where detection was possible within the range of 0.073 to 150 nM. Excellent sensitivity was achieved with the limits of detection of 0.0033, 0.0045, 0.0034, 0.0053 and 0.0048 nM for MC-LR, CYL, anatoxin-α, saxitoxin and OA, respectively. Selectivity studies were performed to show the absence of cross-reactivity between the five analytes. Finally, the application of the multiplexed aptasensor to tap water samples revealed very good agreement with the calibration curves obtained in buffer. This simple and accurate multiplexed platform could open the window for the simultaneous detection of multiple pollutants in different matrices. [ABSTRACT FROM AUTHOR]

MicroRNA (miRNA), crucial non‐coding RNAs, have emerged as key biomarkers in molecular diagnostics, prognosis, and personalized medicine due to their significant role in gene expression regulation. Salivary miRNA, in particular, stands out for its non‐invasive collection method and ease of accessibility, offering promising avenues for the development of point‐of‐care diagnostics for a spectrum of diseases, including cancer, neurodegenerative disorders, and infectious diseases. Such development promises rapid and precise diagnosis, enabling timely treatment. Despite significant advancements in salivary miRNA‐based testing, challenges persist in the quantification, multiplexing, sensitivity, and specificity, particularly for miRNA at low concentrations in complex biological mixtures. This work delves into these challenges, focusing on the development and application of salivary miRNA tests for point‐of‐care use. We explore the biogenesis of salivary miRNA and analyze their quantitative expression and their disease relevance in cancer, infection, and neurodegenerative disorders. We also examined recent progress in miRNA extraction, amplification, and multiplexed detection methods. This study offers a comprehensive view of the development of salivary miRNA‐based point‐of‐care testing (POCT). Its successful advancement could revolutionize the early detection, monitoring, and management of various conditions, enhancing healthcare outcomes. This article is categorized under:Diagnostic Tools > BiosensingDiagnostic Tools > Diagnostic Nanodevices [ABSTRACT FROM AUTHOR]

Pandemics caused by respiratory viruses, such as the SARS-CoV-1/2, influenza virus, and respiratory syncytial virus, have resulted in serious consequences to humans and a large number of deaths. The detection of such respiratory viruses in the early stages of infection can help control diseases by preventing the spread of viruses. However, the diversity of respiratory virus species and subtypes, their rapid antigenic mutations, and the limited viral release during the early stages of infection pose challenges to their detection. This work reports a multiplexed microfluidic immunoassay chip for simultaneous detection of eight respiratory viruses with noticeable infection population, namely, influenza A virus, influenza B virus, respiratory syncytial virus, SARS-CoV-2, human bocavirus, human metapneumovirus, adenovirus, and human parainfluenza viruses. The nanomaterial of the nanozyme (Au@Pt nanoparticles) was optimized to improve labeling efficiency and enhance the detection sensitivity significantly. Nanozyme-binding antibodies were used to detect viral proteins with a limit of detection of 0.1 pg/mL with the naked eye and a microplate reader within 40 min. Furthermore, specific antibodies were screened against the conserved proteins of each virus in the immunoassay, and the clinical sample detection showed high specificity without cross reactivity among the eight pathogens. In addition, the microfluidic chip immunoassay showed high accuracy, as compared with the RT-PCR assay for clinical sample detection, with 97.2%/94.3% positive/negative coincidence rates. This proposed approach thus provides a convenient, rapid, and sensitive method for simultaneous detection of eight respiratory viruses, which is meaningful for the early diagnosis of viral infections. Significantly, it can be widely used to detect pathogens and biomarkers by replacing only the antigen-specific antibodies.

Gliomas are histologically and genetically heterogeneous tumors. However, classical histopathological typing often ignores the high heterogeneity of tumors and thus cannot meet the requirements of precise pathological diagnosis. Here, proximity‐anchored in situ spectral coding amplification (ProxISCA) is proposed for multiplexed imaging of RNA mutations, enabling visual typing of brain gliomas with different pathological grades at the single‐cell and tissue levels. The ligation‐based padlock probe can discriminate one‐nucleotide variations, and the design of proximity primers enables the anchoring of amplicons on target RNA, thus improving localization accuracy. The DNA module‐based spectral coding strategy can dramatically improve the multiplexing capacity for imaging RNA mutations through one‐time labelling, with low cost and simple operation. One‐target‐one‐amplicon amplification confers ProxISCA the ability to quantify RNA mutation copy number with single‐molecule resolution. Based on this approach, it is found that gliomas with higher malignant grades express more genes with high correlation at the cellular and tissue levels and show greater cellular heterogeneity. ProxISCA provides a tool for glioma research and precise diagnosis, which can reveal the relationship between cellular heterogeneity and glioma occurrence or development and assist in pathological prognosis. [ABSTRACT FROM AUTHOR]

In this study, we report a multiplexed platform for the simultaneous determination of five marine toxins. The proposed biosensor is based on a disposable electrical printed (DEP) microarray composed of eight individually addressable carbon electrodes. The electrodeposition of gold nanoparticles on the carbon surface offers high conductivity and enlarges the electroactive area. The immobilization of thiolated aptamers on the AuNP-decorated carbon electrodes provides a stable, well-orientated and organized binary self-assembled monolayer for sensitive and accurate detection. A simple electrochemical multiplexed aptasensor based on AuNPs was designed to synchronously detect multiple cyanotoxins, namely, microcystin-LR (MC-LR), Cylindrospermopsin (CYL), anatoxin-α, saxitoxin and okadaic acid (OA). The choice of the five toxins was based on their widespread presence and toxicity to aquatic ecosystems and humans. Taking advantage of the conformational change of the aptamers upon target binding, cyanotoxin detection was achieved by monitoring the resulting electron transfer increase by square-wave voltammetry. Under the optimal conditions, the linear range of the proposed aptasensor was estimated to be from 0.018 nM to 200 nM for all the toxins, except for MC-LR where detection was possible within the range of 0.073 to 150 nM. Excellent sensitivity was achieved with the limits of detection of 0.0033, 0.0045, 0.0034, 0.0053 and 0.0048 nM for MC-LR, CYL, anatoxin-α, saxitoxin and OA, respectively. Selectivity studies were performed to show the absence of cross-reactivity between the five analytes. Finally, the application of the multiplexed aptasensor to tap water samples revealed very good agreement with the calibration curves obtained in buffer. This simple and accurate multiplexed platform could open the window for the simultaneous detection of multiple pollutants in different matrices.

Magnetic particle spectroscopy (MPS) is an emerging biosensing technique that detects target analytes by exploiting the dynamic magnetic responses of magnetic nanoparticles (MNPs). Due to the ease of synthesis and surface chemical functionalization of MNPs, MPS-based bioassays have gained popularity around the globe. One limiting factor for MPS-based assay is the ability to detect multiple analytes simultaneously in a single run, namely, multiplexed bioassay. Several groups have reported the realization of multiplexed bioassays on surface-based MPS platforms by spatially separating reaction areas by using the unique magnetic responses of different MNPs. In this work, we systematically study the magnetization curves (M-H curves) of different types of MNPs and their relationship to the dynamic magnetic responses when subjected to AC magnetic driving fields. Due to the different structures, sizes, and magnetic properties of each kind of MNP, the resulting harmonics are unique. Thus, concurrent quantification (also called 'colorization') of each type of MNP in a mixture is possible by solving the harmonic matrix function. Our results show that the uniqueness of M-H response curves of selected types of MNP and the signal-to-noise ratio of the system can affect the accuracy of multiplexed, volumetric-based MPS bioassays. The reported method assumes that each type of MNPs nanoparticles does not interact, and that the magnetic response of the mixture is a linear combination of the responses of each kind of MNP. This assumption may not hold for very dense systems where inter-particle interactions become significant and may require more complex models. [ABSTRACT FROM AUTHOR]

Ion‐sensitive field effect transistor (ISFET) sensor is a hot topic these years, playing the combined roles of signal recognizer and converter for (bio)chemical analytes. In this review article, the basic concept, origination, and history of the ISFET sensor are presented. In addition, the common fabrication processes, the most‐used working principle (potentiometric, amperometric, and impedancemetric), and the techniques of gate functionality (physical, chemical, and biological) are discussed introducing the afterward signal transfer processes from ISFET to the terminals through different types of circuits. At last, the development and recent progress (until 2021) of ions and biomolecules (DNA molecules, antibodies, enzymatic substrates, and cell‐related secretions or metabolism) were introduced together with the outlook and facing obstacles (Debye screening, the wearability of ISFET, the multiplexed detections) before the commercialization of ISFET. This review article emphasizes the advantages of the developed ISFET sensors as miniaturization, low‐cost, all‐solid, highly sensitive, and easy operation for portable and multiplexed detections. [ABSTRACT FROM AUTHOR]

Abstract Gliomas are histologically and genetically heterogeneous tumors. However, classical histopathological typing often ignores the high heterogeneity of tumors and thus cannot meet the requirements of precise pathological diagnosis. Here, proximity‐anchored in situ spectral coding amplification (ProxISCA) is proposed for multiplexed imaging of RNA mutations, enabling visual typing of brain gliomas with different pathological grades at the single‐cell and tissue levels. The ligation‐based padlock probe can discriminate one‐nucleotide variations, and the design of proximity primers enables the anchoring of amplicons on target RNA, thus improving localization accuracy. The DNA module‐based spectral coding strategy can dramatically improve the multiplexing capacity for imaging RNA mutations through one‐time labelling, with low cost and simple operation. One‐target‐one‐amplicon amplification confers ProxISCA the ability to quantify RNA mutation copy number with single‐molecule resolution. Based on this approach, it is found that gliomas with higher malignant grades express more genes with high correlation at the cellular and tissue levels and show greater cellular heterogeneity. ProxISCA provides a tool for glioma research and precise diagnosis, which can reveal the relationship between cellular heterogeneity and glioma occurrence or development and assist in pathological prognosis.

Simultaneously studying different biomarker types (DNA, RNA, proteins, etc.) could improve understanding and diagnosis of many complex diseases. However, biomarker detection involves several complex or expensive methodologies, requiring specialized laboratories and personnel. A multiplexed assay would greatly facilitate the use of biomarker data. Here, we present a multiplexed biomarker detection technique using variable‐length DNA payload chains, which are systematically disassembled in the presence of specific biomarkers. The resulting distinctly‐sized fragments yield characteristic gel electrophoresis band patterns. This has enabled us to detect with high sensitivity and specificity DNA sequences including BRCA1 (limit of detection, LOD, ∼3 nM), RNA (miR‐141, LOD ∼19 nM) and the steroids aldosterone and cortisol (LOD ∼200–250 nM). We show that our assay is multiplexable, and suffers limited sensitivity loss in fetal bovine serum and can be applied using capillary electrophoresis, which may be more amenable to automation and integration in healthcare settings. [ABSTRACT FROM AUTHOR]

The early detection at low concentration, by non-invasive methods, of cardiac biomarkers in physiological fluids has attracted the interest of researchers over the last decade. This enables early diagnosis and prediction of the first signs of heart failure (HF). In this respect, the analysis of human saliva remains the most suitable medium for this non-invasive approach, as it contains a highly interesting biological matrix for general health and disease monitoring. In this work, we developed a highly sensitive multiplexed immunosensor for direct simultaneous detection of both N-terminal Natriuretic Peptide (NT-proBNP) and Cortisol in human artificial saliva (AS). The developed biosensor platform based on silicon nitride substrate was composed from four gold working microelectrodes (WEs) and an integrated counter and reference microelectrode. Gold WEs were biofunctionalized through carboxyl diazonium (4-APA) to immobilize both anti-NT-proBNP and anti-Cortisol antibodies for simultaneous detection. The electroaddressing of the 4-APA onto the gold WE surfaces was realized with cyclic voltammetry (CV), while the interaction between antibodies and antigens in PBS was monitored using electrochemical impedance spectroscopy (EIS). The antigen detection in human AS was realized with EIS combined with the standard addition method. The immunosensor was highly sensitive and selective toward the corresponding biomarkers in both PBS and artificial human saliva as well as in the presence of other potential interfering biomarkers such as tumor necrosis factor alpha (TNF-α) and interleukin-10 (IL-10). The limit of detection (LOD) was at 0.2 pg/mL for NT-proBNP within the range of 0.03 to 0.9 pg/mL, while the LOD for Cortisol was 0.06 ng/mL within the range of 0.02 to 0.6 ng/mL for Cortisol in artificial saliva. The developed immunosensor is very promising for significant detection in physiological media, and time reducing as it allows the simultaneous detection of various biomarkers. [ABSTRACT FROM AUTHOR]

Abstract Ion‐sensitive field effect transistor (ISFET) sensor is a hot topic these years, playing the combined roles of signal recognizer and converter for (bio)chemical analytes. In this review article, the basic concept, origination, and history of the ISFET sensor are presented. In addition, the common fabrication processes, the most‐used working principle (potentiometric, amperometric, and impedancemetric), and the techniques of gate functionality (physical, chemical, and biological) are discussed introducing the afterward signal transfer processes from ISFET to the terminals through different types of circuits. At last, the development and recent progress (until 2021) of ions and biomolecules (DNA molecules, antibodies, enzymatic substrates, and cell‐related secretions or metabolism) were introduced together with the outlook and facing obstacles (Debye screening, the wearability of ISFET, the multiplexed detections) before the commercialization of ISFET. This review article emphasizes the advantages of the developed ISFET sensors as miniaturization, low‐cost, all‐solid, highly sensitive, and easy operation for portable and multiplexed detections.

Early detection of cancers and their precise subtyping are essential to patient stratification and effective cancer management. Data-driven identification of expression biomarkers coupled with microfluidics-based detection shows promise to revolutionize cancer diagnosis and prognosis. MicroRNAs play key roles in cancers and afford detection in tissue and liquid biopsies. In this review, we focus on the microfluidics-based detection of miRNA biomarkers in AI-based models for early-stage cancer subtyping and prognosis. We describe various subclasses of miRNA biomarkers that could be useful in machine-based predictive modeling of cancer staging and progression. Strategies for optimizing the feature space of miRNA biomarkers are necessary to obtain a robust signature panel. This is followed by a discussion of the issues in model construction and validation towards producing Software-as-Medical-Devices (SaMDs). Microfluidic devices could facilitate the multiplexed detection of miRNA biomarker panels, and an overview of the different strategies for designing such microfluidic systems is presented here, with an outline of the detection principles used and the corresponding performance measures. Microfluidics-based profiling of miRNAs coupled with SaMD represent high-performance point-of-care solutions that would aid clinical decision-making and pave the way for accessible precision personalized medicine. [ABSTRACT FROM AUTHOR]

The recent progresses in the research of plasmonic phenomena and materials paved the route toward the development of optical sensing platforms based on metal nanostructures with a great potential to be integrated into point-of-care (POC) devices for the next generation of sensing platforms, thus enabling real-time, highly sensitive and accurate diagnostics. In this review, firstly, the optical properties of plasmonic metal nanoparticles will be illustrated, whereafter the engineering of POC platforms, such as microfluidics and readout systems, will be considered with another critical point which is surface functionalization. Attention will also be given to their potential in multiplexed analysis. Finally, the limitations for effective implementation in real diagnostics will be illustrated with a special emphasis on the latest trend in developing cutting-edge sensing systems. [ABSTRACT FROM AUTHOR]

High-throughput protein assays are crucial for modern diagnostics, drug discovery, proteomics, and other fields of biology and medicine. It allows simultaneous detection of hundreds of analytes and miniaturization of both fabrication and analytical procedures. Photonic crystal surface mode (PC SM) imaging is an effective alternative to surface plasmon resonance (SPR) imaging used in conventional gold-coated, label-free biosensors. PC SM imaging is advantageous as a quick, label-free, and reproducible technique for multiplexed analysis of biomolecular interactions. PC SM sensors are characterized by a longer signal propagation at the cost of a lower spatial resolution, which makes them more sensitive than classical SPR imaging sensors. We describe an approach for designing label-free protein biosensing assays employing PC SM imaging in the microfluidic mode. Label-free, real-time detection of PC SM imaging biosensors using two-dimensional imaging of binding events has been designed to study arrays of model proteins (antibodies, immunoglobulin G-binding proteins, serum proteins, and DNA repair proteins) at 96 points prepared by automated spotting. The data prove feasibility of simultaneous PC SM imaging of multiple protein interactions. The results pave the way to further develop PC SM imaging as an advanced label-free microfluidic assay for the multiplexed detection of protein interactions. [ABSTRACT FROM AUTHOR]

Respiratory tract infections such as the ongoing coronavirus disease 2019 (COVID-19) has seriously threatened public health in the last decades. The experience of fighting against the epidemic highlights the importance of user-friendly and accessible point-of-care systems for nucleic acid (NA) detection. To realize low-cost and multiplexed point-of-care NA detection, a swing-assisted multiplexed analyzer for point-of-care respiratory tract infection testing (SMART) was proposed to detect multiple respiratory tract pathogens using visible loop-mediated isothermal amplification. By performing hand-swing movements to generate acceleration force to distribute samples into reaction chambers, the design of the SMART system was greatly simplified. By using different format of chips and integrating into a suitcase, this system can be applied to on-site multitarget and multi-sample testing. Three targets including the N and Orf genes of SARS-CoV-2 and the internal control were simultaneously analyzed (limit of detection: 2000 copies/mL for raw sample; 200 copies/mL for extracted sample). Twenty-three clinical samples with eight types of respiratory bacteria and twelve COVID-19 clinical samples were successfully detected. These results indicate that the SMART system has the potential to be further developed as a versatile tool in the diagnosis of respiratory tract infection. [ABSTRACT FROM AUTHOR]

Metal-organic frameworks (MOFs)-based optical nanoprobes for luminescence and surface-enhanced Raman spectroscopy (SERS) applications have been receiving tremendous attention. Every element in the MOF structure, including the metal nodes, the organic linkers, and the guest molecules, can be used as a source to build single/multi-emission signals for the intended analytical purposes. For SERS applications, the MOF can not only be used directly as a SERS substrate, but can also improve the stability and reproducibility of the metal-based substrates. Additionally, the porosity and large specific surface area give MOF a sieving effect and target molecule enrichment ability, both of which are helpful for improving detection selectivity and sensitivity. This mini-review summarizes the advances of MOF-based optical detection methods, including luminescence and SERS, and also provides perspectives on future efforts. [ABSTRACT FROM AUTHOR]

Circulating tumor DNA (ctDNA) is a critical biomarker not only important for the early detection of tumors but also invaluable for personalized treatments. Currently ctDNA detection relies on sequencing. Here, a platform termed three-dimensional-coded interlocked DNA rings (3D-coded ID rings) was created for multiplexed ctDNA identification. The ID rings provide a ctDNA recognition ring that is physically interlocked with a reporter ring. The specific binding of ctDNA to the recognition ring initiates target-responsive cutting via a restriction endonuclease; the cutting then triggers rolling circle amplification on the reporter ring. The signals are further integrated with internal 3D codes for multiplexed readouts. ctDNAs from non-invasive clinical specimens including plasma, feces, and urine were detected and validated at a sensitivity much higher than those obtained through sequencing. This 3D-coded ID ring platform can detect any multiple DNA fragments simultaneously without sequencing. We envision that our platform will facilitate the implementation of future personalized/precision medicine.

An automated microfluidic system with functionalized beads has been developed for multiplexed TORCH detection at point-of-care testing. A concise microfluidic chip consisting of a one-dimensional beads array is developed to simultaneously detect TOX, RUB, CMV, HSV-I and HSV-II respectively with five functionalized beads. A compact liquid handling module has been developed to automate the sandwiched chemiluminescence immunoassay within the one-dimensional beads array of the microfluidic chip. A precise ram pump is adopted to not only add reagent into the microfluidic chip from outside, but also facilitate elaborate fluid control inside the microfluidic chip for improved performance. A large-size waste chamber with a liquid-absorbing sponge holds the waste reagent within the microfluidic chip to prevent backflow. The one-dimensional beads array is heated from double-sides at 37 ℃ for sensitive detection with reduced time. A sensitive CMOS camera is adopted to take chemiluminescence image from the one-dimensional beads array, and a custom processing algorithm is adopted to analyze the image. For each serum sample, five different infections can be simultaneously detected with the automated microfluidic system. Experimental results show that efficient, sensitive, and accurate multiplexed TORCH detection can be conveniently achieved with the integrated microfluidic system. [ABSTRACT FROM AUTHOR]

In this article, we present the development of a volatile organic compound (VOC) gas sensor based on graphite on a printed circuit board (PCB) multiplexed interdigitated electrode (IDE) sensor. We report the effects of graphite as a sensing material deposited by the spray method and its interaction response to specific gases. In addition, we demonstrate how we designed and manufactured IDE sensors with an integrated heating circuit and thermistor element, as well as the 55- $\mu \text{L}$ microchamber, which contributed to the fast detection of gases. All experiments were performed with four IDE sensors to verify the reproducibility and sensitivity of the proposed methodology. Our results highlight that acetone presented the highest detection sensitivity, with an electric current increase of ± $100~\mu \text{A}$ at a 200-ppm cycle with a response of 0.92 s and full recovery at 2.62 s. Furthermore, the developed sensors were able to detect and distinguish between acetone, methanol, isopropanol, ethanol, and chloroform gases. The sensors exhibited outstanding stability and repeatability when evaluated after six months. The sensors’ operation at 2 V makes it suitable for low-power devices with a high potential to be applied in future commercial room-temperature VOC gas sensor devices. [ABSTRACT FROM AUTHOR]

In this review, we present the current trends in photonic biosensors, focusing on devices based on lab-on-a-chip (LOC) systems capable of simultaneously detecting multiple real-life diseases on a single platform. The first section lists the advantages and challenges of building LOC platforms based on integrated optics. Some of the most popular materials for the fabrication of microfluidic cells are also shown. Then, a review of the latest developments in biosensors using the evanescent wave detection principle is provided; this includes interferometric biosensors, ring resonators, and photonic crystals, including a brief description of commercial solutions, if available. Then, a review of the latest advances in surface plasmon resonance (SPR) biosensors is presented, including localized-SPRs (LSPRs). A brief comparison between the benefits and required improvements on each kind of biosensor is discussed at the end of each section. Finally, prospects in the field of LOC biosensors based on integrated optics are glimpsed. [ABSTRACT FROM AUTHOR]

A polymer nanostructured electrodes-enabled electrochemical sensing microchip for detecting neurotransmitters is reported. This microchip can detect neurotransmitters with a level as low as ∼120 nM with high specificity in both static and dynamic conditions. Real-time monitoring of dopamine released from N27-A dopaminergic neural cells under potassium chloride excitation using the microchip has been demonstrated. [Display omitted] • Neurotransmitters are used to transmit messages between neurons. • Abnormal levels of the neurotransmitters lead to neurological disorders. • Nanostructured electrochemical sensing chip offers a cost-effective and sensitive method for detecting neurotransmitters. • Real-time monitoring of neurotransmitter released from dopaminergic neural cells has been demonstrated. Neurotransmitters are used by the nervous system to transmit messages between neurons. The abnormal levels of the neurotransmitters may lead to neurological disorders. It is very important to monitor their levels in patients. Herein, we report a polymer nanostructured electrodes-enabled electrochemical sensing microchip for detecting dopamine and serotonin. The nanostructures on the electrode can enhance the surface area of the electrode dramatically. As a result, the measured electrical signals increased in comparison with those of an electrochemical sensor with an electrode of a flat surface. It has been found that this microchip can detect neurotransmitters with a level as low as ∼ 120 nM with high specificity and can be used to monitor the dopamine and serotonin in a mixed sample successfully in both static and dynamic conditions. Finally, the real-time measurements of dopamine released from N27-A dopaminergic neural cells using the microchip have been demonstrated. [ABSTRACT FROM AUTHOR]

[Display omitted] • A new detachable microneedle platform has been developed to access the ISF and monitoring of key biomarkers. • The platform can be customized by pairing it with different test papers to fulfill specific sensing requirements. • The design of the biosensor features a mortise and tenon structure, significantly improving its durability and versatility. • The sampling performance of the platform has good stability and robustness. The microneedle array provides a painless method for penetrating the skin to reach interstitial fluid (ISF), making it suitable for drug delivery and monitoring of key biomarkers. While current microneedle sensors integrate sensitive components for detecting specific biomarkers, challenges remain in their development, particularly in diversifying detection capabilities and ensuring durability. To address these challenges, we present a detachable microneedle platform capable of monitoring various biomolecules through colorimetric and electrochemical sensing. This platform is also compatible with the Urit uric acid tester, which, when coupled with the microneedles (MNs), demonstrates satisfactory stability results. The design features a mortise-tenon structure that significantly enhances the durability of the biosensor, while the provision of insertable cavities increases its versatility. By pairing with a variety of test strips, the platform can be customized to meet specific sensing requirements for a range of metabolites, aiding in disease management and regulation. [ABSTRACT FROM AUTHOR]

The rapid and precise detection of pathogenic agents is critical for public health and societal stability. The detection of biological warfare agents (BWAs) is especially vital within military and counter-terrorism contexts, essential in defending against biological threats. Traditional methods, such as polymerase chain reaction (PCR), are limited by their need for specific settings, impacting their adaptability and versatility. This study introduces a cell-free biosensor for BWA detection by converting the 16S rRNA of targeted pathogens into detectable functional protein molecules. The modular nature of this approach allows for the flexible configuration of pathogen detection, enabling the simultaneous identification of multiple pathogenic 16S rRNAs through customized reporter proteins for each targeted sequence. Furthermore, we demonstrate how this method integrates with techniques utilizing retroreflective Janus particles (RJPs) for facile and highly sensitive pathogen detection. The cell-free biosensor, employing RJPs to measure the reflection of non-chromatic white light, can detect 16S rRNA from BWAs at femtomolar levels, corresponding to tens of colony-forming units per milliliter of pathogenic bacteria. These findings represent a significant advancement in pathogen detection, offering a more efficient and accessible alternative to conventional methodologies. [ABSTRACT FROM AUTHOR]

Electrochemiluminescence (ECL) is a bioanalytical technique with numerous advantages, including the potential for high temporal and spatial resolution, a high signal-to-noise ratio, a broad dynamic range, and rapid measurement capabilities. To reduce the complexity of a multi-electrode approach, we use a single-electrode electrochemiluminescence (SE-ECL) configuration to achieve the simultaneous emission and detection of multiple colors for applications that require multiplexed detection of several analytes. This method exploits intrinsic differences in the electric potential applied along single electrodes built into electrochemical cells, enabling the achievement of distinct colors through selective excitation of ECL luminophores. We present results on the optimization of SE-ECL intensity for different channel lengths and widths, with sum intensities being 5 times larger for 6 cm vs. 2 cm channels and linearly increasing with the width of the channels. Furthermore, we demonstrated for the first time that applying Alternating Current (AC) voltage within the single electrode setup for driving the ECL reactions has a dramatic effect on the emitted light intensity, with square waveforms resulting in higher intensities vs sine waveforms. Additionally, multiplexed multicolor SE-ECL on a 6.5 mm × 3.6 mm CMOS semiconductor image sensor was demonstrated for the first time, with the ability to simultaneously distinguish four different colors, leading to the ability to measure multiple analytes. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC), as one of the most lethal cancers, significantly impacts human health. Attempts in this area tends to develop novel technologies with sensitive and multiplexed detection properties for early diagnosis. Here, we present novel hydrogel photonic crystal (PhC) barcodes with tyramine deposition amplified enzyme-linked immunosorbent assay (ELISA) for highly sensitive and multiplexed HCC biomarker screening. Because of the abundant amino groups of acrylic acid (AA) component, the constructed hydrogel PhC barcodes with inverse opal structure could facilitate the loading of antibody probes for subsequent detection of tumor markers. By integrating tyramine deposition amplified ELISA on the barcode, the detection signal of tumor markers has been enhanced. Based on these features, it is demonstrated that the hydrogel PhC barcodes with tyramine deposition amplified ELISA could realize highly sensitive and multiplexed detection of HCC-related biomarkers. It was found that this method is flexible, sensitive and accurate, suitable for multivariate analysis of low abundance tumor markers and future cancer diagnosis. These features make the newly developed PhC barcodes an innovation platform, which possesses tremendous potential for practical application of low abundance targets. [ABSTRACT FROM AUTHOR]

The early detection at low concentration, by non-invasive methods, of cardiac biomarkers in physiological fluids has attracted the interest of researchers over the last decade. This enables early diagnosis and prediction of the first signs of heart failure (HF). In this respect, the analysis of human saliva remains the most suitable medium for this non-invasive approach, as it contains a highly interesting biological matrix for general health and disease monitoring. In this work, we developed a highly sensitive multiplexed immunosensor for direct simultaneous detection of both N-terminal Natriuretic Peptide (NT-proBNP) and Cortisol in human artificial saliva (AS). The developed biosensor platform based on silicon nitride substrate was composed from four gold working microelectrodes (WEs) and an integrated counter and reference microelectrode. Gold WEs were biofunctionalized through carboxyl diazonium (4-APA) to immobilize both anti-NT-proBNP and anti-Cortisol antibodies for simultaneous detection. The electroaddressing of the 4-APA onto the gold WE surfaces was realized with cyclic voltammetry (CV), while the interaction between antibodies and antigens in PBS was monitored using electrochemical impedance spectroscopy (EIS). The antigen detection in human AS was realized with EIS combined with the standard addition method. The immunosensor was highly sensitive and selective toward the corresponding biomarkers in both PBS and artificial human saliva as well as in the presence of other potential interfering biomarkers such as tumor necrosis factor alpha (TNF-α) and interleukin-10 (IL-10). The limit of detection (LOD) was at 0.2 pg/mL for NT-proBNP within the range of 0.03 to 0.9 pg/mL, while the LOD for Cortisol was 0.06 ng/mL within the range of 0.02 to 0.6 ng/mL for Cortisol in artificial saliva. The developed immunosensor is very promising for significant detection in physiological media, and time reducing as it allows the simultaneous detection of various biomarkers.

Molecular diagnostics have significantly advanced the early detection of diseases, where the electrochemical sensing of biomarkers (e.g., DNA, RNA, proteins) using multiple electrode arrays (MEAs) has shown considerable promise. Nanostructuring the electrode surface results in higher surface coverage of capture probes and more favorable orientation, as well as transport phenomena unique to nanoscale, ultimately leading to enhanced sensor performance. The central goal of this study is to investigate the influence of electrode nanostructure on electrically-guided immobilization of DNA probes for nucleic acid detection in a multiplexed format. To that end, we used nanoporous gold (np-Au) electrodes that reduced the limit of detection (LOD) for DNA targets by two orders of magnitude compared to their planar counterparts, where the LOD was further improved by an additional order of magnitude after reducing the electrode diameter. The reduced electrode diameter also made it possible to create a np-Au MEA encapsulated in a microfluidic channel. The electro-grafting reduced the necessary incubation time to immobilize DNA probes into the porous electrodes down to 10 min (25-fold reduction compared to passive immobilization) and allowed for grafting a different DNA probe sequence onto each electrode in the array. The resulting platform was successfully used for the multiplexed detection of three different biomarker genes relevant to breast cancer diagnosis.

Multiplexed detection is essential in biomedical sciences since it is more efficient and accurate than single-analyte detection. For an accurate early diagnosis of COVID-19, a multiplexed detection strategy is required to avoid false negatives with the existing gold standard assay. Nb2CTx nanosheets were found to efficiently quench the fluorescence emission of lanthanide-doped upconversion luminescence nanoparticles at wavelengths ranging from visible to near-infrared spectrum. Using this broad-spectrum quencher, we developed a label-free FRET-based biosensor for rapid and accurate detection of SARS-CoV-2 RNA. To target ORF and N genes, two types of oligo-modified lanthanide-doped upconversion nanoparticles can be used simultaneously to identify-two sites in one assay via upconversion fluorescence enhancement intensity measurement with detection limits of 15 pM and 914 pM, respectively. Moreover, with multisite cross-validation, this multiplexed and sensitive biosensor is capable of simultaneous and multicolor analysis of two gene fragments of SARS-CoV-2 Omicron variant within minutes in a single homogeneous solution, which significantly improves the detection efficiency. The diagnosis result via our assay is consistent with the PCR result, demonstrating its application in the rapid and accurate screening of multiple genes of SARS-CoV-2 and other infectious diseases.

Sepsis is a silent killer, caused by a syndromic reaction of the body's immune system to an infection that is typically the ultimate pathway to mortality due to numerous infectious diseases, including COVID‐19 across the world. In the United States alone, sepsis claims 220,000 lives, with a dangerously high fatality rate between 25% and 50%. Early detection and treatment can avert 80% of sepsis mortality which is currently unavailable in most healthcare institutions. The novelty in this work is the ability to simultaneously detect eight (IL‐6, IL‐8, IL‐10, IP‐10, TRAIL, d‐dimer, CRP, and G‐CSF) heterogeneous immune response biomarkers directly in whole blood without the need for dilution or sample processing. The DETecT sepsis (Direct Electrochemical Technique Targeting Sepsis) 2.0 sensor device leverages electrochemical impedance spectroscopy as a technique to detect subtle binding interactions at the metal/semi‐conductor sensor interface and reports results within 5 min using only two drops (~100 μl) of blood. The device positively (r >0.87) correlated with lab reference standard LUMINEX for clinical translation using 40 patient samples. The developed device showed diagnostic accuracy greater than 80% (AUC >0.8) establishing excellent specific and sensitive response. Portable handheld user‐friendly feature coupled with precise quantification of immune biomarkers makes the device amenable in a versatile setting providing insights on patient's immune response. This work highlights an innovative solution of enhancing sepsis care and management in the absence of a decision support device in the continuum of sepsis care. [ABSTRACT FROM AUTHOR]

Many emerging technologies have the potential to improve health care by providing more personalized approaches or early diagnostic methods. In this review, we cover smartphone-based multiplexed sensors as affordable and portable sensing platforms for point-of-care devices. Multiplexing has been gaining attention recently for clinical diagnosis considering certain diseases require analysis of complex biological networks instead of single-marker analysis. Smartphones offer tremendous possibilities for on-site detection analysis due to their portability, high accessibility, fast sample processing, and robust imaging capabilities. Straightforward digital analysis and convenient user interfaces support networked health care systems and individualized health monitoring. Detailed biomarker profiling provides fast and accurate analysis for disease diagnosis for limited sample volume collection. Here, multiplexed smartphone-based assays with optical and electrochemical components are covered. Possible wireless or wired communication actuators and portable and wearable sensing integration for various sensing applications are discussed. The crucial features and the weaknesses of these devices are critically evaluated. [ABSTRACT FROM AUTHOR]

Immunoassay is one of the most important clinical techniques for disease/pathological diagnosis. Mass spectrometry (MS) has been a popular and powerful readout technique for immunoassays, generating the mass spectrometric immunoassays (MSIAs) with unbeatable channels for multiplexed detection. The sensitivity of MSIAs has been greatly improved with the development of mass labels from element labels to small-molecular labels. MSIAs are also expended from the representative element MS-based methods to the laser-based organic MS and latest ambient MS, improving in both technology and methodology. Various MSIAs present high potential for clinical applications, including the biomarker screening, the immunohistochemistry, and the advanced single-cell analysis. Here, we give an overall review of the development of MSIAs in recent years, highlighting the latest improvement of mass labels and MS techniques for clinical immunoassays. [ABSTRACT FROM AUTHOR]

A major challenge for ultrasensitive analysis is the high-efficiency determination of different target single molecules in parallel with high accuracy. Herein, we developed a quantitative fluoro-electrochemical imaging approach for direct multiplexed single-molecule counting with a SiC-nanofilm-modified indium tin oxide transparent electrode. The nanofilm could control local pH through proton-coupled electron transfer in a lower potential range and further induce direct electrochemical oxidation of the dye molecules with a higher applied potential. The fluoro-electrochemical responses of immobilized single molecules with different pH values and redox behaviors could thus be distinguished within the same fluorescence channels. This method yields nonamplified direct counting of single molecules, as indicated by excellent linear responses in the picomolar range. The successful distinction of seven different randomly mixed dyes underscores the versatility and efficacy of the proposed method in the highly accurate determination of single dye molecules, paving the way for highly parallel single-molecule detection for diverse applications.

The COVID-19 pandemic, in addition to the co-occurrence of influenza virus and respiratory syncytial virus (RSV), has emphasized the requirement for efficient and reliable multiplex diagnostic methods for respiratory infections. While existing multiplex detection techniques are based on reverse transcription quantitative polymerase chain reaction (RT-qPCR) and extraction and purification kits, the need for complex instrumentation and elevated cost limit their scalability and availability. In this study, we have developed a point-of-care (POC) device based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) that can simultaneously detect four respiratory viruses (SARS-CoV-2, Influenza A, Influenza B, and RSV) and perform two controls in less than 30 min, while avoiding the use of the RNA extraction kit. The system includes a disposable microfluidic cartridge with mechanical components that automate sample processing, with a low-cost and portable optical reader and a smartphone app to record and analyze fluorescent images. The application as a real point-of-care platform was validated using swabs spiked with virus particles in nasal fluid. Our portable diagnostic system accurately detects viral RNA specific to respiratory pathogens, enabling deconvolution of coinfection information. The detection limits for each virus were determined using virus particles spiked in chemical lysis buffer. Our POC device has the potential to be adapted for the detection of new pathogens and a wide range of viruses by modifying the primer sequences. This work highlights an alternative approach for multiple respiratory virus diagnostics that is well-suited for healthcare systems in resource-limited settings or at home.

Abstract Sepsis is a silent killer, caused by a syndromic reaction of the body's immune system to an infection that is typically the ultimate pathway to mortality due to numerous infectious diseases, including COVID‐19 across the world. In the United States alone, sepsis claims 220,000 lives, with a dangerously high fatality rate between 25% and 50%. Early detection and treatment can avert 80% of sepsis mortality which is currently unavailable in most healthcare institutions. The novelty in this work is the ability to simultaneously detect eight (IL‐6, IL‐8, IL‐10, IP‐10, TRAIL, d‐dimer, CRP, and G‐CSF) heterogeneous immune response biomarkers directly in whole blood without the need for dilution or sample processing. The DETecT sepsis (Direct Electrochemical Technique Targeting Sepsis) 2.0 sensor device leverages electrochemical impedance spectroscopy as a technique to detect subtle binding interactions at the metal/semi‐conductor sensor interface and reports results within 5 min using only two drops (~100 μl) of blood. The device positively (r >0.87) correlated with lab reference standard LUMINEX for clinical translation using 40 patient samples. The developed device showed diagnostic accuracy greater than 80% (AUC >0.8) establishing excellent specific and sensitive response. Portable handheld user‐friendly feature coupled with precise quantification of immune biomarkers makes the device amenable in a versatile setting providing insights on patient's immune response. This work highlights an innovative solution of enhancing sepsis care and management in the absence of a decision support device in the continuum of sepsis care.

The recent progresses in the research of plasmonic phenomena and materials paved the route toward the development of optical sensing platforms based on metal nanostructures with a great potential to be integrated into point-of-care (POC) devices for the next generation of sensing platforms, thus enabling real-time, highly sensitive and accurate diagnostics. In this review, firstly, the optical properties of plasmonic metal nanoparticles will be illustrated, whereafter the engineering of POC platforms, such as microfluidics and readout systems, will be considered with another critical point which is surface functionalization. Attention will also be given to their potential in multiplexed analysis. Finally, the limitations for effective implementation in real diagnostics will be illustrated with a special emphasis on the latest trend in developing cutting-edge sensing systems.

Simultaneous detection of different biomarkers from a single specimen in a single test, allowing more rapid, efficient, and low-cost analysis, is of great significance for accurate diagnosis of disease and efficient monitoring of therapy. Recently, developments in microfabrication and nanotechnology have advanced the integration of nanomaterials in microfluidic devices toward multiplex assays of biomarkers, combining both the advantages of microfluidics and the unique properties of nanomaterials. In this review, we focus on the state of the art in multiplexed detection of biomarkers based on nanomaterial-assisted microfluidics. Following an overview of the typical microfluidic analytical techniques and the most commonly used nanomaterials for biochemistry analysis, we highlight in detail the nanomaterial-assisted microfluidic strategies for different biomarkers. These highly integrated platforms with minimum sample consumption, high sensitivity and specificity, low detection limit, enhanced signals, and reduced detection time have been extensively applied in various domains and show great potential in future point-of-care testing and clinical diagnostics. [ABSTRACT FROM AUTHOR]

The similar transmission patterns and early symptoms of respiratory viral infections, particularly severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza (H1N1), and respiratory syncytial virus (RSV), pose substantial challenges in the diagnosis, therapeutic management, and handling of these infectious diseases. Multiplexed point-of-care testing for detection is urgently needed for prompt and efficient disease management. Here, we introduce an electrochemical paper-based analytical device (ePAD) platform for multiplexed and label-free detection of SARS-CoV-2, H1N1, and RSV infection using immobilized pyrrolidinyl peptide nucleic acid probes. Hybridization between the probes and viral nucleic acid targets causes changes in the electrochemical response. The resulting sensor offers high sensitivity and low detection limits of 0.12, 0.35, and 0.36 pM for SARS-CoV-2 (N gene), H1N1, and RSV, respectively, without showing any cross-reactivities. The amplification-free detection of extracted RNA from 42 nasopharyngeal swab samples was successfully demonstrated and validated against reverse-transcription polymerase chain reaction (range of cycle threshold values: 17.43–25.89). The proposed platform showed excellent clinical sensitivity (100 %) and specificity (≥97 %) to achieve excellent agreement (κ ≥ 0.914) with the standard assay, thereby demonstrating its applicability for the screening and diagnosis of these respiratory diseases. [Display omitted] • The PNA-based ePAD was developed for multiplex detection of respiratory viruses. • This platform can be used to diagnose SARS-CoV-2, H1N1, and RSV infection. • The developed device achieves high specificity and sensitivity for the detection. • This system can be applied to clinical samples without requiring RNA amplification. • The analysis results corresponded well with those of the RT-PCR assays. [ABSTRACT FROM AUTHOR]

With significant advancements in understanding gene functions and therapy, the potential misuse of gene technologies, particularly in the context of sports through gene doping (GD), has come to the forefront. This raises concerns regarding the need for point-of-care testing of various GD candidates to counter illicit practices in sports. However, current GD detection techniques, such as PCR, lack the portability required for on-site multiplexed detection. In this study, we introduce an integrated microfluidics-based chip for multiplexed gene doping detection, termed MGD-Chip. Through the strategic design of hydrophilic and hydrophobic channels, MGD-Chip enables the RPA and CRISPR-Cas12a assays to be sequentially performed on the device, ensuring minimal interference and cross-contamination. Six potential GD candidates were selected and successfully tested simultaneously on the platform within 1 h. Demonstrating exceptional specificity, the platform achieved a detection sensitivity of 0.1 nM for unamplified target plasmids and 1 aM for amplified ones. Validation using mouse models established by injecting IGFI and EPO transgenes confirmed the platform's efficacy in detecting gene doping in real samples. This technology, capable of detecting multiple targets using portable elements, holds promise for real-time GD detection at sports events, offering a rapid, highly sensitive, and user-friendly solution to uphold the integrity of sports competitions. [ABSTRACT FROM AUTHOR]

Disease progression of sepsis has been perceived as a multifaceted phenomenon, considering the temporal host inflammatory response within individuals which requires early diagnosis. Herein, we present a multicohort analysis through temporal inflammatory biomarker profiling using Direct Electrochemical Technique Targeting (DETecT) sepsis device that measures and quantify cytokines (IL-6, IL-8, IL-10), chemokines (TRAIL, IP-10), and well-established inflammatory biomarkers (PCT, CRP) with a sample turnaround time of 0.97) with the Luminex reference standard during clinical evaluation for a total of 124 sepsis patient samples. Low mean bias for all the biomarkers in Bland- Altman analysis indicated good agreement between the standard LUMINEX method and the developed DETecT sepsis device. We used the combinatorial power of rapidly measuring a panel of seven biomarkers, paired with a machine learning model, to effectively predict the patient outcomes when given two-time points in the early stages of sepsis. The device could predict patient mortality and recovery with over 92% accuracy by applying decision tree analysis. We envision this work would facilitate personalized treatment based on biomarker stratification to represent exactly where the patient belongs within the sepsis continuum. Measurable empirical data with a fast turnaround time would facilitate the DETecT sepsis device as a potential enabling technology that can play a crucial role in understanding sepsis prognosis and be leveraged for personalized therapeutics anywhere.

In this study, a homologous multi-wavelength fluorescence polarization immunoassays (MWFPIA) for rapid determination of amantadine (AMD) and ribavirin (RVB) simultaneously was established. The MWFPIA could quantify AMD and RVB concentration simultaneously in 1 min. After optimisation, long-wavelength tracers AEDA-AF647 with monoclonal antibody (Mab) 1A12 and RVB-CP-EDF with polyclonal antibody (Pab) G412 were chosen for the development of MWFPIA. The limit of detections (LODs) of AMD and RVB were 1.7 and 1.0 μg/kg in chickens, and 17.6 and 10.4 μg/L in human serum, respectively. Recoveries of AMD and RVB in chickens were 61.2–94.9% and 87.3–90.2%, respectively with coefficient of variations (CVs) lower than 16.7%. For human serum, recoveries were 60.2–84.3% and 63.5–74.2% for AMD and RVB, respectively, and CVs was lower than 15.3%. The results indicated that the MWFPIA could be used for the monitoring of antiviral drugs with short analysis time, great accuracy and high throughput.

The multiplexed detection of metabolites in parallel within a single biosensor plate is sufficiently valuable but also challenging. Herein, we combine the inherent light addressability of silicon with the high selectivity of enzymes, for the construction of multiplexed photoelectrochemical enzymatic biosensors. To conduct a stable electrochemistry and reagentless biosensing on silicon, a new strategy involving the immobilization of both redox mediators and enzymes using an amide bond-based hydrogel membrane was proposed. The membrane characterization results demonstrated a covalent coupling of ferrocene mediator to hydrogel, in which the mediator acted as not only a signal generator but also a renewable sacrifice agent. By adding corresponding enzymes on different spots of hydrogel membrane modified silicon and recording local photocurrents with a moveable light pointer, this biosensor setup was used successfully to detect multiple metabolites, such as lactate, glucose, and sarcosine, with good analytical performances. The limits of detection of glucose, sarcosine and lactate were found to be 179 μM, 16 μM, and 780 μM with the linear ranges of 0.5–2.5 mM, 0.3–1.5 mM, and 1.0–3.0 mM, respectively. We believe this proof-of-concept study provides a simple and rapid one-step immobilization approach for the fabrication of reagentless enzymatic assays with silicon-based light-addressable electrochemistry. [Display omitted] [ABSTRACT FROM AUTHOR]

[Display omitted] • A framework nucleic acids (FNAs) fluorescent nanoprobe was constructed. • The detection of multiple DNA repair enzymes in living cells was realized. • This method exhibits good selectivity and high sensitivity. • The FNAs enter the cell autonomously and image UDG and APE1. DNA repair enzymes play pivotal roles in DNA damage repair. Imaging multiple DNA repair enzymes in living cells is significant for DNA damage repair-related biological study and disease diagnosis. Here, we constructed a framework nucleic acids (FNAs) fluorescent nanoprobe for imaging multiple DNA repair enzymes in living cells. Uracil DNA glycosylase (UDG) and human apurinic/apyrimidinic endonuclease 1 (APE1) were used as the target analytes. Two double-stranded DNA probes (dsDNA1 and dsDNA2) were designed. dsDNA1 contained four uracil (U) bases for UDG recognition and was labeled with black hole quencher2 (BHQ2) /cyanine5 (Cy5). dsDNA2 contained one apurinic/apyrimidinic (AP) site for APE1 recognition and was labeled with black hole quencher1 (BHQ1) /fluorophore carboxyfluorescein (FAM). To obtain the FNAs fluorescent nanoprobe, dsDNA1 and dsDNA2 were attached to two vertices of a DNA tetrahedron (a type of FNAs). Under the action of UDG and APE1, dsDNA1 and dsDNA2 were both dissociated, generating fluorescence of Cy5 and FAM. Among them, Cy5 indicated the activity of UDG and FAM indicated the activity of APE1. This method had good sensitivity to UDG and APE1, and the detection limits were 0.0012 U/mL and 0.057 U/mL, respectively. This method also exhibited high selectivity for UDG and APE1. When the FNAs fluorescent nanoprobe was endocytosed, it had little toxicity to normal and cancer cells. Furthermore, this method successfully achieved multiple imaging UDG and APE1 in cancer cells. This study provides a novel strategy for imaging multiple DNA repair enzymes and holds great potential in biological applications and clinical diagnosis. [ABSTRACT FROM AUTHOR]

High-throughput protein assays are crucial for modern diagnostics, drug discovery, proteomics, and other fields of biology and medicine. It allows simultaneous detection of hundreds of analytes and miniaturization of both fabrication and analytical procedures. Photonic crystal surface mode (PC SM) imaging is an effective alternative to surface plasmon resonance (SPR) imaging used in conventional gold-coated, label-free biosensors. PC SM imaging is advantageous as a quick, label-free, and reproducible technique for multiplexed analysis of biomolecular interactions. PC SM sensors are characterized by a longer signal propagation at the cost of a lower spatial resolution, which makes them more sensitive than classical SPR imaging sensors. We describe an approach for designing label-free protein biosensing assays employing PC SM imaging in the microfluidic mode. Label-free, real-time detection of PC SM imaging biosensors using two-dimensional imaging of binding events has been designed to study arrays of model proteins (antibodies, immunoglobulin G-binding proteins, serum proteins, and DNA repair proteins) at 96 points prepared by automated spotting. The data prove feasibility of simultaneous PC SM imaging of multiple protein interactions. The results pave the way to further develop PC SM imaging as an advanced label-free microfluidic assay for the multiplexed detection of protein interactions.

Respiratory tract infections such as the ongoing coronavirus disease 2019 (COVID-19) has seriously threatened public health in the last decades. The experience of fighting against the epidemic highlights the importance of user-friendly and accessible point-of-care systems for nucleic acid (NA) detection. To realize low-cost and multiplexed point-of-care NA detection, a swing-assisted multiplexed analyzer for point-of-care respiratory tract infection testing (SMART) was proposed to detect multiple respiratory tract pathogens using visible loop-mediated isothermal amplification. By performing hand-swing movements to generate acceleration force to distribute samples into reaction chambers, the design of the SMART system was greatly simplified. By using different format of chips and integrating into a suitcase, this system can be applied to on-site multitarget and multi-sample testing. Three targets including the N and Orf genes of SARS-CoV-2 and the internal control were simultaneously analyzed (limit of detection: 2000 copies/mL for raw sample; 200 copies/mL for extracted sample). Twenty-three clinical samples with eight types of respiratory bacteria and twelve COVID-19 clinical samples were successfully detected. These results indicate that the SMART system has the potential to be further developed as a versatile tool in the diagnosis of respiratory tract infection.

Metal-organic frameworks (MOFs)-based optical nanoprobes for luminescence and surface-enhanced Raman spectroscopy (SERS) applications have been receiving tremendous attention. Every element in the MOF structure, including the metal nodes, the organic linkers, and the guest molecules, can be used as a source to build single/multi-emission signals for the intended analytical purposes. For SERS applications, the MOF can not only be used directly as a SERS substrate, but can also improve the stability and reproducibility of the metal-based substrates. Additionally, the porosity and large specific surface area give MOF a sieving effect and target molecule enrichment ability, both of which are helpful for improving detection selectivity and sensitivity. This mini-review summarizes the advances of MOF-based optical detection methods, including luminescence and SERS, and also provides perspectives on future efforts.