Multiplexing Capability Research Articles

SAMBA: A Multicolor Digital Melting PCR Platform for Rapid Microbiome Profiling (Small Methods 8/2022)

Rapid Microbiome Profiling In article number 2200185, Cheow and co-workers showed that melt curve analysis of multicolor sloppy molecular beacon enables significant expansion of the multiplexing capability in digital polymerase chain reaction.

Ultrasensitive and rapid detection of artemisinin based on bismuth tungstate dressed rGO nanocomposite

Sensor based on nanomaterials act as a new player because of multiplexing capability, specificity and sensitivity. Here, bismuth tungstate dressed reduced graphene oxide (rGO); composite material is fabricated and demonstrated its application for the ultra-sensitive detection of artemisinin. Nano flowers shaped microspheres morphology was revealed through scanning electron microscopes with increased surface roughness. Conducting nanoparticles exhibit great potential of applications in conduction based-nanosensors. Novel bismuth tungstate dressed rGO nanocomposite was synthesized, attributed for artemisinin detection. The new unique morphology of composite enabled us to use it in pharmaceutical sensing with enhanced sensitivity and efficiency. To study the multiple sensing parameters of designed material; multiple modes of PGSTAT (DPV, amperometric and cyclic voltammetry) were used to record the responses. The designed material bismuth tungstate dressed rGO act as recognition material for artemisinin and limit of detection was found as 0.01 μM. Such unique nanomaterials pave a way to fabricate cost effective, ultrasensitive and super selective detection methods.

Programmable Analysis of MicroRNAs by Thermus thermophilus Argonaute-Assisted Exponential Isothermal Amplification for Multiplex Detection (TEAM).

The simultaneous analysis of the levels of multiple microRNAs (miRNAs) is critical to the early diagnosis of cancer. However, this analysis is challenging because of the low concentrations of miRNAs and their high sequence homology. Here, we report a general and programmable diagnostic strategy for miRNA analysis: Thermus thermophilus Argonaute (TtAgo)-assisted exponential isothermal amplification for multiplex detection (TEAM). This system combines exponential isothermal amplification (EXPAR), for target amplification, with programmable TtAgo cleavage, for the generation of the reporting signal. The TEAM assay achieved attomolar sensitivity with a rapid turnaround time (30-35 min). Because of the single-nucleotide precision of TtAgo, the system demonstrated robust multiplex capability in the simultaneous detection of four miRNA targets and the classification of let-7 family members. The TEAM assay was superior in differentiating colorectal cancer patients from healthy individuals relative to the conventional EXPAR and reverse transcription polymerase chain reaction (RT-PCR) methods. This tunable and scalable approach is a powerful nucleic acid analysis tool that holds promise in scientific and clinical applications.

Asymmetric Mach-Zehnder Interferometric Biosensing for Quantitative and Sensitive Multiplex Detection of Anti-SARS-CoV-2 Antibodies in Human Plasma.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic has once more emphasized the urgent need for accurate and fast point-of-care (POC) diagnostics for outbreak control and prevention. The main challenge in the development of POC in vitro diagnostics (IVD) is to combine a short time to result with a high sensitivity, and to keep the testing cost-effective. In this respect, sensors based on photonic integrated circuits (PICs) may offer advantages as they have features such as a high analytical sensitivity, capability for multiplexing, ease of miniaturization, and the potential for high-volume manufacturing. One special type of PIC sensor is the asymmetric Mach–Zehnder Interferometer (aMZI), which is characterized by a high and tunable analytical sensitivity. The current work describes the application of an aMZI-based biosensor platform for sensitive and multiplex detection of anti-SARS-CoV-2 antibodies in human plasma samples using the spike protein (SP), the receptor-binding domain (RBD), and the nucleocapsid protein (NP) as target antigens. The results are in good agreement with several CE-IVD marked reference methods and demonstrate the potential of the aMZI biosensor technology for further development into a photonic IVD platform.

Plasmonic Fluor-Enhanced Antigen Arrays for High-Throughput, Serological Studies of SARS-CoV-2.

Serological testing for acute infection or prior exposure is critical for patient management and coordination of public health decisions during outbreaks. Current methods have several limitations, including variable performance, relatively low analytical and clinical sensitivity, and poor detection due to antigenic drift. Serological methods for SARS-CoV-2 detection for the ongoing COVID-19 pandemic suffer from several of these limitations and serves as a reminder of the critical need for new technologies. Here, we describe the use of ultrabright fluorescent reagents, Plasmonic Fluors, coupled with antigen arrays that address a subset of these limitations. We demonstrate its application using patient samples in SARS-CoV-2 serological assays. In our multiplexed assay, SARS-CoV-2 antigens were spotted into 48-plex arrays within a single well of a 96-well plate and used to evaluate remnant laboratory samples of SARS-CoV-2 positive patients. Signal-readout was performed with Auragent Bioscience’s Empower microplate reader, and microarray analysis software. Sample volumes of 1 μL were used. High sensitivity of the Plasmonic Fluors combined with the array format enabled us to profile patient serological response to eight distinct SARS-CoV-2 antigens and evaluate responses to IgG, IgM, and IgA. Sensitivities for SARS-CoV-2 antigens during the symptomatic state ranged between 72.5 and 95.0%, specificity between 62.5 and 100%, and the resulting area under the curve values between 0.76 and 0.97. Together, these results highlight the increased sensitivity for low sample volumes and multiplex capability. These characteristics make Plasmonic Fluor-enhanced antigen arrays an attractive technology for serological studies for the COVID-19 pandemic and beyond.

Operando Decoding of Surface Strain in Anode-Free Lithium Metal Batteries via Optical Fiber Sensor.

With zero excess lithium, anode‐free lithium metal batteries (AFLMBs) can deliver much higher energy density than that of traditional lithium metal batteries. However, AFLMBs are prone to suffer from rapid capacity loss and short life. Monitoring and analyzing the capacity decay of AFLMBs are of great importance for their future applications. It is known that the capacity fade mainly comes from the formation of solid electrolyte interphase species and dead lithium, which leads to irreversible volume expansion. Therefore, monitoring and distinguishing the irreversible volume expansion or reversible volume expansion are the key points to analyze the capacity fade of AFLMBs. Herein, an applicable technique based on optical fiber sensors to characterize and quantize the volume change of AFLMBs is developed. By attaching fiber Bragg grating (FBG) sensors onto the surface of the multilayered anode‐free pouch cells, the strain evolution of the cells is successfully monitored and correlated with their electrochemical properties. It is found that the decline of surface strain fluctuation amplitude caused by the loss of active lithium is the leading indicator of battery failure. The proposed sensing technique has excellent multiplexing capability that can be considered as an elementary unit for capacity fade analysis in next‐generation battery management system.

The Mini Colon Model: a benchtop multi-bioreactor system to investigate the gut microbiome

ABSTRACT In vitro fermentation systems allow for the investigation of gut microbial communities with precise control of various physiological parameters while decoupling confounding factors from the human host. Current systems, such as the SHIME and Robogut, are large in footprint, lack multiplexing, and have low experimental throughput. Alternatives which address these shortcomings, such as the Mini Bioreactor Array system, are often reliant on expensive specialized equipment, which hinders wide replication across labs. Here, we present the Mini Colon Model (MiCoMo), a low-cost, benchtop multi-bioreactor system that simulates the human colon environment with physiologically relevant conditions. The device consists of triplicate bioreactors working independently of an anaerobic chamber and equipped with automated pH, temperature, and fluidic control. We conducted 14-d experiments and found that MiCoMo was able to support a stable complex microbiota community with a Shannon Index of 3.17 ± 0.65, from individual fecal samples after only 3–5 d of inoculation. MiCoMo also retained inter-sample microbial differences by developing closely related communities unique to each donor, while maintaining both minimal variations between replicate reactors (average Bray-Curtis similarity 0.72 ± 0.13) andday-to-day variations (average Bray-Curtis similarity 0.81±0.10) after this short stabilization period. Together, these results establish MiCoMo as an accessible system for studying gut microbial communities with high throughput and multiplexing capabilities.

Novel Lateral Flow-Based Assay for Simple and Visual Detection of SARS-CoV-2 Mutations.

Identification of the main SARS-CoV-2 variants in real time is of interest to control the virus and to rapidly devise appropriate public health responses. The RT-qPCR is currently considered to be the reference method to screen SARS-CoV-2 mutations, but it has some limitations. The multiplexing capability is limited when the number of markers to detect increases. Moreover, the performance of this allele-specific method may be impacted in the presence of new mutations. Herein, we present a proof-of-concept study of a simple molecular assay to detect key SARS-CoV-2 mutations. The innovative features of the assay are the multiplex asymmetric one-step RT-PCR amplification covering different regions of SARS-CoV-2 S gene and the visual detection of mutations on a lateral flow DNA microarray. Three kits (Kit 1: N501Y, E484K; Kit 2: L452R, E484K/Q; Kit 3: K417N, L452R, E484K/Q/A) were developed to match recommendations for surveillance of SARS-CoV-2 variants between January and December 2021. The clinical performance was assessed using RNA extracts from 113 SARS-CoV-2-positive samples with cycle thresholds <30, and results demonstrated that our assay allows specific and sensitive detection of mutations, with a performance comparable to that of RT-qPCR. The VAR-CoV assay detected four SARS-CoV-2 targets and achieved specific and sensitive screening of spike mutations associated with the main variants of concern, with a performance comparable to that of RT-qPCR. With well-defined virus sequences, this assay can be rapidly adapted to other emerging mutations; it is a promising tool for variant surveillance.

Scalable multiplex co-fractionation/mass spectrometry platform for accelerated protein interactome discovery

Co-fractionation/mass spectrometry (CF/MS) enables the mapping of endogenous macromolecular networks on a proteome scale, but current methods are experimentally laborious, resource intensive and afford lesser quantitative accuracy. Here, we present a technically efficient, cost-effective and reproducible multiplex CF/MS (mCF/MS) platform for measuring and comparing, simultaneously, multi-protein assemblies across different experimental samples at a rate that is up to an order of magnitude faster than previous approaches. We apply mCF/MS to map the protein interaction landscape of non-transformed mammary epithelia versus breast cancer cells in parallel, revealing large-scale differences in protein-protein interactions and the relative abundance of associated macromolecules connected with cancer-related pathways and altered cellular processes. The integration of multiplexing capability within an optimized workflow renders mCF/MS as a powerful tool for systematically exploring physical interaction networks in a comparative manner.

Paper-Based Fluidic Sensing Platforms for β-Adrenergic Agonist Residue Point-of-Care Testing

The illegal use of β-adrenergic agonists during livestock growth poses a threat to public health; the long-term intake of this medication can cause serious physiological side effects and even death. Therefore, rapid detection methods for β-adrenergic agonist residues on-site are required. Traditional detection methods such as liquid chromatography have limitations in terms of expensive instruments and complex operations. In contrast, paper methods are low cost, ubiquitous, and portable, which has led to them becoming the preferred detection method in recent years. Various paper-based fluidic devices have been developed to detect β-adrenergic agonist residues, including lateral flow immunoassays (LFAs) and microfluidic paper-based analytical devices (μPADs). In this review, the application of LFAs for the detection of β-agonists is summarized comprehensively, focusing on the latest advances in novel labeling and detection strategies. The use of μPADs as an analytical platform has attracted interest over the past decade due to their unique advantages and application for detecting β-adrenergic agonists, which are introduced here. Vertical flow immunoassays are also discussed for their shorter assay time and stronger multiplexing capabilities compared with LFAs. Furthermore, the development direction and prospects for the commercialization of paper-based devices are considered, shedding light on the development of point-of-care testing devices for β-adrenergic agonist residue detection.

A direct and multiplex digital PCR chip for EGFR mutation

Digital PCR is a sensitive detection method, which has important applicability in liquid biopsy through the measurement of ctDNA. However, the current sample pre-processing of ctDNA and the multiplex detection capability of digital PCR have limitations. In view of the above two aspects, we developed a digital PCR chip with multiplex capability and established a direct amplification detection method without nucleic acid extraction. Through the design and processing of the chip, we established a self-priming multiplex digital PCR chip, which can detect 4 targets using single fluorescence. This method can be applied to most digital PCR chips. In addition, we used the plasma of lung cancer patients to establish a direct digital PCR detection method based on the chip, thereby avoiding disadvantages caused by the ctDNA extraction process. As a proof of concept, we prepared blood plasma samples with different concentration of ctDNA to prove the chip's multiplex detection capabilities and the results suggested that this multiplex digital PCR is accurate. Overall, our platform provides a novel and promising option for the detection of ctDNA.

Metasurface-based THz reflectarray antenna with vortex multiplexing and beam-steering capabilities for future wireless communications

SummaryThis article investigates the unexplored potentials of vortices or orbital angular momentum (OAM) beams using the low-cost and high-gain dielectric reflectarray antennas (RAs) at the terahertz (THz) band. It proposes a paradigm to enable 3D beam-steering or OAM multiplexing by a single structure via tilted OAM beams. That, in turn, requires reaching the maximal attainable angles either to send multiple beams to different receivers or to focus the OAM beams of different modes in a desired direction. For this reason, two concepts are addressed in this work: (i). generating a single 3D steered OAM beam and (ii) producing multiple off-centered OAM beams with different modes. A volumetric unit cell is adopted to be accurately tuned through the aperture to steer the generated beams towards the desired direction(s). The proposed paradigm can be utilized to produce RAs with beam-steering or OAM multiplexing capabilities as candidates for THz indoor communications.

Peptidomic Approaches and Observations in Neurodegenerative Diseases.

Mass spectrometry (MS), with its immense technological developments over the last two decades, has emerged as an unavoidable technique in analyzing biomolecules such as proteins and peptides. Its multiplexing capability and explorative approach make it a valuable tool for analyzing complex clinical samples concerning biomarker research and investigating pathophysiological mechanisms. Peptides regulate various biological processes, and several of them play a critical role in many disease-related pathological conditions. One important example in neurodegenerative diseases is the accumulation of amyloid-beta peptides (Aβ) in the brain of Alzheimer’s disease (AD) patients. When investigating brain function and brain-related pathologies, such as neurodegenerative diseases, cerebrospinal fluid (CSF) represents the most suitable sample because of its direct contact with the brain. In this review, we evaluate publications applying peptidomics analysis to CSF samples, focusing on neurodegenerative diseases. We describe the methodology of peptidomics analysis and give an overview of the achievements of CSF peptidomics over the years. Finally, publications reporting peptides regulated in AD are discussed.

16-ports indoor base station MIMO array for sub-6 GHz 5G applications

A typical 5G multiple-input and multiple-output (MIMO) system must combine a high number of antennas at both the transmitter and receiver to realize spatial multiplexing capability. In this paper, a wideband 16- element indoor base station (BS) antenna array that can cover 3.3–6.0 GHz is proposed for 5G applications. A π-shaped monopole antenna is designed to cover the Lower band (LTE bands 42/43–N77–N78), the intermediate band (N79), and the higher band (LTE 46). The antenna elements are arranged in a limited space printed on a substrate take the Hexakaidecagon Polygon shape. As the antenna elements are arranged with good isolation, achieving good polarization diversity. The proposed BS array is simulated, fabricated, and tested. The typical results, S-parameters, antenna efficiency, and radiation patterns are investigated. Moreover, to validate the MIMO performances, a very low envelope correlation coefficient (ECC) below 0.02, high antenna efficiency of about 82–93.2% are achieved. The calculated ergodic channel capacity of the 16 × 16 MIMO system reached up to 85 bps/Hz. The proposed antenna array was compared to some other 5G MIMO indoor base station antennas.

Recent advances in isobaric labeling and applications in quantitative proteomics.

Mass spectrometry (MS) has emerged at the forefront of quantitative proteomic techniques. Liquid chromatography-mass spectrometry (LC-MS) can be used to determine abundances of proteins and peptides in complex biological samples. Several methods have been developed and adapted for accurate quantification based on chemical isotopic labeling. Among various chemical isotopic labeling techniques, isobaric tagging approaches rely on the analysis of peptides from MS2-based quantification rather than MS1-based quantification. In this review, we will provide an overview of several isobaric tags along with some recent developments including complementary ion tags, improvements in sensitive quantitation of analytes with lower abundance, strategies to increase multiplexing capabilities, and targeted analysis strategies. We will also discuss limitations of isobaric tags and approaches to alleviate these restrictions through bioinformatic tools and data acquisition methods. This review will highlight several applications of isobaric tags, including biomarker discovery and validation, thermal proteome profiling, cross-linking for structural investigations, single-cell analysis, top-down proteomics, along with applications to different molecules including neuropeptides, glycans, metabolites, and lipids, while providing considerations and evaluations to each application.

Force-Coded Strategy for the Simultaneous Detection of Multiple Tumor-Related Proteins.

Multiplexed simultaneous detection of various cancer markers is required for accurate diagnosis and treatment of early cancer. In this work, we present a force-coded strategy for the simultaneous detection of tumor-related proteins with tunable dynamic range via magnetic sensing. The multiplexing capability of this method is achieved by designing DNA devices that can recognize different biomarkers and code them with different binding forces measured by the force-induced remnant magnetization spectroscopy, which is not influenced by the color of the light and the solution. Moreover, the force-coded assay with high sensitivity and adjustable detection range is robust, which could be used for practical biological applications such as magnetic sensing, handheld miniaturized systems, and potential in vivo diagnosis.

Dynamic Single-Molecule Sensors: A Theoretical Study.

One of the key advantages of single-molecule sensors over conventional ensemble technologies is their capability of revealing the heterogeneity among molecular events. In dynamic single-molecule sensing, heterogeneity in molecular interaction kinetics is quantified as the fingerprint to specifically detect target molecules. This strategy offers a unique approach to develop ultrasensitive biosensors with a limit of detection at the fM level, which is three orders of magnitude lower than that of conventional assays. However, due to the lack of a comprehensive theoretical model, the rational design of dynamic single-molecule sensors is challenging. Herein, we present the theoretical study of sensing performance with a hydrodynamic model. We quantitatively show that there is a dilemma regarding the probe design. High-affinity probes offer higher specificity but require extremely long assay time, while low-affinity probes could shorten the assay time but are prone to the interference from unwanted molecules. This study also suggests that one possible solution to solve this dilemma is by applying external disturbance to the system, as we have recently demonstrated by experiments. We anticipate that this work could inspire the rational design of single-molecule sensors to further improve the sensitivity, specificity, and multiplexing capability.

Abstract 2457: PICASSO: Ultra-multiplexed imaging technique for spatial protein analysis of tumor microenvironments

Abstract In recent cancer research in medicine, various combinatorial biomarkers are simultaneously visualized to study molecular heterogeneity of the tumor microenvironment in detail. These kinds of study aim to provide enhanced precise diagnostic system, optimal treatment decision, and more accurate prediction of survival or recurrence rates for each independent patient case. Conventional immunofluorescence-based approach, however, has been faced an intrinsic limitation in terms of spectral overlap among fluorophores, which allows only 3-4 fluorescent channels per single imaging cycle. Various alternatives, including mass cytometry or linear unmixing, have been proposed to overcome and enhance the multiplexing capability, while they could not totally replace the conventional immunofluorescence technique due to its requirement of specialized equipment and high barrier to entry. Here we report a state-of-the-art multiplexed imaging technique, termed PICASSO, which can perform an unprecedented high multiplexing capability even with a conventional microscopy platform. PICASSO is mainly composed of three parts; 1. antibody complex preformation technique for multiple target staining without host issue, 2. acquiring spectrally mixed images at different spectral ranges, in which each channel included one of those fluorophores’ emission peaks, indicating the number of image acquisitions equal to the number of fluorophores, and 3. fluorescent signals unmixing via mutual information (MI) minimization algorithm. Fifteen-color multiplexed imaging has been experimentally demonstrated within a single staining and imaging cycle via PICASSO. As a cyclic staining method, we demonstrated that PICASSO enables more than 40-color imaging within 3 staining and imaging rounds. Moreover, we demonstrated multiplexed imaging of mouse brains using commercialized bandpass filter-based microscopy. Other than mouse brain samples, various formalin-fixed paraffin-embedded (FFPE) specimens (e.g., breast, kidney, bladder, colon, prostate, thymus, etc.) could be utilized for PICASSO unmixing demonstration, which infers the feasibility of PICASSO technique in various pathological studies in medicine. In addition, PICASSO can be applied to 3D multiplexed imaging, mRNA multiplexed imaging, and large-area multiplexed imaging. We anticipate that PICASSO will provide a new perspective in multiplexed imaging field with high accessibility in cancer research and promote how we see and understand the molecular profiles of tumors and their microenvironments. Acknowledgments: This work was supported by Samsung Research Funding &amp; Incubation Center for Future Technology (SRFC-IT1702-09). Citation Format: Junyoung Seo, Yeonbo Sim, Jeewon Kim, Hyunwoo Kim, In Cho, Hoyeon Nam, Young-Gyu Yoon, Jae-Byum Chang. PICASSO: Ultra-multiplexed imaging technique for spatial protein analysis of tumor microenvironments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2457.

A CDM-WDM Interrogation Scheme for Massive Serial FBG Sensor Networks

Structural health monitoring and other smart structures gain an increased attention which can be satisfied by the quasi-distributed optical sensing approach. Serial fiber optic sensors, such as fiber-Bragg gratings (FBGs) provide among others small size, immunity to electro-magnetic interference, an accurate sensing accuracy and a high multiplexing capability to increase the amount of sensing points in an optical sensor network. Different multiplexing approaches demonstrated a limited number of FBGs, such as time-division multiplex, optical frequency domain refractometry or frequency shifted interferometry. This work introduces a code-division multiplex (CDM) - wavelength-division multiplex (WDM) interrogator for massive serial FBG sensor networks. The interrogation of 4000 serial sensors in a network with a length of 113m, 200 identical WDM sections over a length of 200m and a network with 1000 sensors and a length of 1.6km show a massive multiplexing capability of up to 16000 sensors and possible network lengths of several kilometers. Strain measurements with FBGs in rear sections prove the sensing applicability of the CDM-WDM scheme.

Abstract 2932: The 6-color naica® system: a digital PCR platform and workflow for high multiplex genetic detection and analysis

Abstract There are several molecular platforms routinely used to investigate genomic material. Quantitative polymerase chain reaction (qPCR) is ubiquitously used because of the ease of use and the precision however, it does not offer high multiplexing. While digital PCR (dPCR) is known for increased precision and sensitivity, most digital PCR platforms provide the same level of multiplexing as qPCR. Next-generation sequencing (NGS) is less precise than qPCR or dPCR and but has the highest multiplexing capability of the three technologies. Conventional digital PCR systems have been limited by the number of instruments required to conduct the workflow, time-to-result compared to qPCR, and level of multiplexing as compared to next-generation sequencing (NGS). Crystal Digital PCR™ and the naica system is a next-generation digital PCR platform that simplifies the number of instruments in the workflow, speeds up the time of results to less than 3 hours, and increases the number of detection colors to 6 spectrally distinct channels. By using a combination of dual-labelled fluorescent probes, multiplexed digital PCR provides a simple solution that makes it possible to use less of a precious sample when faced with limited quantity without sacrificing sensitivity. The naica system workflow utilizes microfluidic consumables, such as the Sapphire chip for high sensitivity applications, and the Opal chip for higher throughput studies. All reagents including primers and probes, master mix, and sample are inputted into the chips in one single loading step. Chips are transferred to the Geode instrument, where the sample is partitioned into a 2D-monolayer of droplet crystals and where all targets are amplified. After the PCR program has been completed, the same chips are then loaded into the Prism6 imager which detects all targets across 6 fluorescent channels and outputs an image of all droplets in a sample. Finally, Crystal Miner analysis software can be used to analyze and decipher the quantifications and expression of all targets in the assay. The 6-color naica system allows for high multiplex detection of targets previously unattainable by conventional digital PCR systems. With this advent, genomic scientists have an additional platform in the molecular toolbox that supports seamless assay development of oncology biomarker assays for gene expression, rare target detection, and liquid biopsy applications. Citation Format: Andrew Hoskins, Shekerah Primus, Mary Yazdani, Kimberley Gutierrez, Benjamin Foret, Fabien Bernard, Rémi Dangla. The 6-color naica® system: a digital PCR platform and workflow for high multiplex genetic detection and analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2932.