Multiplexing Capacity Research Articles

Optical multiple-image compression-encryption via single-pixel Radon transform.

Compression is a common operation in multiple-image encryption (MIE) schemes. For most of the existing MIE schemes, the compression process would lead to the waste of the detect data. In this paper, a novel MIE scheme based on the single-pixel Radon transform (RT) is proposed. In the encryption process, the single-pixel RT of each image from 0° to 180° is performed. This process can be realized by an optical setup. All Radon spectra are combined and encrypted by the asymmetric cryptosystem based on phase-truncated Fourier transforms (PTFT). To increase the security, the Arnold map and logistic map are used to scramble and diffuse the result of PTFT. Unlike conventional MIE schemes, there is no extra compression operation and data waste in the proposed method. The key sensitivity, the multiplexing capacity, and the security under various attacks are analyzed through simulation results. The optical experiment is also performed to demonstrate the feasibility of the proposed optical MIE scheme.

12-Plex UHPLC-MS/MS analysis of sarcosine in human urine using integrated principle of multiplex tags chemical isotope labeling and selective imprint enriching

Urinary sarcosine was considered to be a potential biomarker for prostate cancer (Pca). In this work, an integrated strategy of multiplex tags chemical isotope labeling (MTCIL) combined with magnetic dispersive solid phase extraction (MDSPE), was proposed for specific extraction and high-throughput determination of sarcosine by ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). In the past three months, we have developed 8-plex MTCIL reagents with excellent qualitative and quantitative performance. In this work, the multiplexing capacity of MTCIL reagents (MTCIL360/361/362/363/364/365/366/375/376/378/379/381) was increased 1.5-fold from 8-plex to 12-plex. MTCIL359 was prepared and used to label sarcosine standard as internal standard (IS). The structural analogue derivative (MTCIL373-sarcosine) of all targeted MTCIL-sarcosine derivatives was synthesized and used as a novel dummy template to prepare dummy magnetic molecularly imprinted polymers (DMMIPs). The integration of MTCIL and DMMIPs procedures were extremely favorable to excellent chromatographic separation and efficient mass spectrometric detection. The labeling efficiency, chromatographic retention and mass spectrometry responses of MTCIL reagents were consistent for sarcosine. In a single UHPLC-MS/MS run (2.0 min), this method can simultaneously quantify sarcosine in 12-plex urine samples and achieve unbiased concentrations comparison between different urine samples. Analytical parameters including linearity (R2 0.989–0.997), detection limits (0.02 nM), precision (2.6–11.5%), accuracy (96.1–107.4%), matrix effect, labeling and extraction efficiency were carefully validated. The proposed method was successfully applied for urinary sarcosine determination of healthy male individuals and Pca patients. It was found that the sarcosine concentrations in these two groups were statistically extremely significantly different (P < 0.001). The developed method was a powerful analytical tool to substantially promote the analysis throughput and large-scale experiments about the potential biomarker research.

Ultra-sensitive and rapid detection of nucleic acids and microorganisms in body fluids using single-molecule tethering

Detection of microbial nucleic acids in body fluids has become the preferred method for rapid diagnosis of many infectious diseases. However, culture-based diagnostics that are time-consuming remain the gold standard approach in certain cases, such as sepsis. New culture-free methods are urgently needed. Here, we describe Single MOLecule Tethering or SMOLT, an amplification-free and purification-free molecular assay that can detect microorganisms in body fluids with high sensitivity without the need of culturing. The signal of SMOLT is generated by the displacement of micron-size beads tethered by DNA probes that are between 1 and 7 microns long. The molecular extension of thousands of DNA probes is determined with sub-micron precision using a robust and rapid optical approach. We demonstrate that SMOLT can detect nucleic acids directly in blood, urine and sputum at sub-femtomolar concentrations, and microorganisms in blood at 1 CFU mL−1 (colony forming unit per milliliter) threefold faster, with higher multiplexing capacity and with a more straight-forward protocol than amplified methodologies. SMOLT’s clinical utility is further demonstrated by developing a multiplex assay for simultaneous detection of sepsis-causing Candida species directly in whole blood.

High-throughput and Deep-proteome Profiling by 16-plex Tandem Mass Tag Labeling Coupled with Two-dimensional Chromatography and Mass Spectrometry.

Isobaric tandem mass tag (TMT) labeling is widely used in proteomics because of its high multiplexing capacity and deep proteome coverage. Recently, an expanded 16-plex TMT method has been introduced, which further increases the throughput of proteomic studies. In this manuscript, we present an optimized protocol for 16-plex TMT-based deep-proteome profiling, including protein sample preparation, enzymatic digestion, TMT labeling reaction, two-dimensional reverse-phase liquid chromatography (LC/LC) fractionation, tandem mass spectrometry (MS/MS), and computational data processing. The crucial quality control steps and improvements in the process specific for the 16-plex TMT analysis are highlighted. This multiplexed process offers a powerful tool for profiling a variety of complex samples such as cells, tissues, and clinical specimens. More than 10,000 proteins and posttranslational modifications such as phosphorylation, methylation, acetylation, and ubiquitination in highly complex biological samples from up to 16 different samples can be quantified in a single experiment, providing a potent tool for basic and clinical research.

Abstract 1352: Enhanced target capturing throughput and simplified workflow via innovative chemistry on hybrid capture blockers

Abstract Hybrid capture is a popular and well-accepted method to select targeted genomic regions for subsequent next generation sequencing (NGS). Despite its popularity in research and clinical diagnostics, hybrid capture NGS is now facing the challenge by the high throughput demands generated by Novaseq, Illumina's flagship platform that occupies the research and industry market rapidly in recent years. Novaseq create tetrabytes of sequencing data per run, allowing unprecedented level of sample multiplexing, especially for targeted NGS. Conventional blockers, whose sample index-matching region uses either degenerate bases or universal pairing bases, still need to match the length of the sample index for each individual library. When using NovaSeq, scientists need to combine huge number of samples using different types of index system in each single run. However, matching different hybridization blockers with corresponding index system is time consuming and error-prone. Here we introduce a novel design and chemistry to hybrid capture blockers to maximized the pooling capacity of hybrid capture to match the increasing need of sample throughput and multiplexing. Our new hybrid capture blockers are not only an essential component in the target enrichment workflow to block the permissive interactions between library adapters and target probes, which may lower the capturing efficiency and generate excessive data from the off-target sequence, but also significantly promotes the capturing capacity and efficiency, and simplifies the workflow. Our new blocker, GenFisher Universal Blockers, using our proprietary design and chemistry, create a novel solution to the conjugation between sample indexes on libraries and blockers, enabling blockers bind adapters regardless to the index length and sequence. In our experiment, NGS libraries are created with a commercially available kit and TruSeq style adapters that contain sample indexes with 3 different lengths: 6nt, 8nt and 10nt. Hybrid captures were then carried out with 8,000-probe GenFisher hybridization panel and kit and Universal Blockers for each individual libraries and the combined library with different indexes. Sequencing data showed that the on-target base percentage was consistently at &amp;lt;75% for all captures for both individual or combined multiplex libraries, whereas hybrid capture without blockers yielded &amp;gt;30% on-target rate. We also compared GenFisher Universal Blockers with the blockers from two other industrial leading providers, which are also claimed to be ‘universal'. Our data showed that GenFisher Universal Blockers was the only truly universal solution that worked with all 3 index lengths unbiasedly while the other two are still index length-sensitive and struggled with 10nt indexes library capturing (on-target ~50%, compared to &amp;lt;65% at 6/8nt). With its versatility, GenFisher Universal Blockers enables higher multiplexing capacity, as well as the freedom of index selection to hybrid capture NGS users. Citation Format: Jianpeng Wang, Long Fan, Lumeng Ye, Jiashi Wang, Irene Song, Cedric Wu. Enhanced target capturing throughput and simplified workflow via innovative chemistry on hybrid capture blockers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1352.

Abstract 5540: Digital nanoneedle biosensors enable comprehensive monitoring of systemic inflammatory response to immunotherapies

Abstract Cytokine release syndrome (CRS), a systemic inflammatory response observed in some monoclonal antibody drugs and adaptive T-Cell therapies has become a major issue in cancer immunotherapy. CRS can present as a mild reaction requiring minimally invasive supportive care up to a severe systemic response resulting in patient death. Monitoring this response during these therapeutic treatments is non-trivial due to the wide range of biomarker concentrations, small sample volumes, and long assay times. CRS-associated biomarkers including CRP and ferritin vary from 10ng/mL-10mg/mL while other biomarkers (eg, IL6, IFNγ, etc.) vary from 1pg/mL-100ng/mL. At present, no analytical tool, known to us, can provide this large dynamic range simultaneously with the requisite low limit of detection in a single test. NanoMosaic technology has the required sensitivity and dynamic range for the quantification of a large panel of CRS biomarkers in a single assay. The NanoMosaic technology is powered by silicon nanoneedle biosensors that are densely integrated on a plate and manufactured with CMOS-compatible nanofabrication processes. Each nanoneedle is a label-free biosensor, functionalized with capture antibodies, and changes its scattering spectrum when an antigen binds to its surface. Each analyte specific sensing area consists a total of ~24k nanoneedles divided into a digital region (~20k nanoneedles), an analog region (~3k nanoneedles) and a fabrication QC region (~1k nanoneedles). The colors of all nanoneedles are imaged in a single shot with a low-cost color camera. The digital nanoneedles provide the single molecule sensitivity. Therefore, at ultra-low concentration when antigens captured by the nanoneedles follow Poisson statistics, the number of antigens can be quantitated by counting the presence or absence of color changes of individual nanoneedles in a binary fashion. As the protein concentrations increase, the positive counts increase accordingly and achieve saturation when all nanoneedles capture more than one protein. Above the saturation concentration, an adjacent section of analog nanoneedles perform quantitative analysis based on the level of color change, thus providing a wider dynamic range beyond the digital counting concentration ranges. Each single analyte area, including both digital and analog sensors, is less than 500um. Therefore, high level multiplex can be achieved by parallelizing the detection in a microarray format, while maintaining the same level of sensitivity and dynamic range. We have developed various assays on the NanoMosaic platform, including cytokines such as TNF-alpha, IL6, CRP, IFNγ, as well as low-abundance proteins such as Tau proteins at 50fg/mL, in different matrices. Prognostic monitoring of the inflammatory immune response requires simultaneous measurement of multiple cytokines at widely divergent concentrations. NanoMosaic technology provides the sensitivity, dynamic range and multiplexing capacity required to fully integrate patient immune response into therapeutic development and decision making. Citation Format: Qimin Quan, Joshua Ritchey, Mark Clenow, Joe Wilkinson, John Geanacopoulos, John Boyce. Digital nanoneedle biosensors enable comprehensive monitoring of systemic inflammatory response to immunotherapies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5540.

Fiber optic sensors enabled monitoring of thermal curling of concrete pavement slab: Temperature, strain and inclination

An intellectual system is developed to monitor concrete pavement slab subjected to external temperature fluctuations. Truly distributed fiber-optic temperature and strain sensors and an innovative Fabry-Perot interferometer-based optical fiber inclinometer are installed in the concrete pavement slab so as to monitor and evaluate its thermal curling process. The system is demonstrated to be effective in detecting the thermal curling of slab. The results substantiate that the top surface of the slab has a fast reaction rate to the heating/cooling process. The bottom surface temperature has a time-lag comparing to the top surface, which results in a sharp increase then a slow reduction in the top-vs-bottom temperature difference. In the light of the inclinometer, it is proven that the tilt angle of the curvature of the slab has a high degree of synchronization with the top-vs-bottom temperature difference. The temperature/strain distribution maps are also presented and uneven distributions of temperature and strain are captured. Moreover, taking the advantage of the multiplexing capacity of the distributed fiber-optic sensors, the coefficient of thermal expansion (CTE) of the slab can be easily obtained and used to feed a finite element model. Finally, a three-dimensional finite element model is established to completely understand the curling process measured from the monitoring system. The model and the experimental data agree well with each other. While the sensors output realistic temperature and deformations at certain locations, the validated model is able to reveal more details of the nonlinearity inside the slab.

A Comparative Study of Asynchronous and Synchronous OCDMA Systems

This article presents a detailed survey on both asynchronous and synchronous Optical Code-Division Multiple-Access (OCDMA) systems based on 1-D, 2-D, and 3-D codes, distinctively. Accordingly, different coding schemes along with their multiplexing capacity, correlation features, advantages and drawbacks are addressed and benchmarked. Additionally, different detection techniques such as direct and coherent detection and their capability to handle Multiple Access-Interference are also discussed. Furthermore, the main OCDMA systems from the last two decades are summarized considering their coding schemes, performance, device components, and applications. Therefore, a new comparative study in terms of spectral efficiency and bit error rate for 1-D, 2-D, and 3-D asynchronous and synchronous OCDMA systems is carried out. Numerical results shown that the 3-D MD code stands out among the codes with superior performance as well as support to higher data rates and number of users.

Monitoring of inflammatory response to cancer immunotherapies.

e15199 Background: Inflammation observed in response to some monoclonal antibody drugs and adaptive T-Cell therapies has become a major issue in cancer immunotherapy. Prognostic monitoring of the inflammatory response requires simultaneous measurement of multiple cytokines at widely divergent concentrations. At present, no analytical method, known to us, can provide large dynamic range (&gt; 6 logs), high sensitivity (&lt; 1pg/ml) and high multiplex in a single test. Methods: The NanoMosaic platform is a cytokine quantification technology powered by silicon nanoneedle biosensors that are densely integrated on a plate and manufactured with CMOS-compatible nanofabrication processes. Each nanoneedle is a label-free biosensor, functionalized with capture antibodies. Each analyte specific sensing area consists a total of ~23k nanoneedles divided into a digital region (~20k nanoneedles) and an analog region (~3k nanoneedles), combined to cover the entire range of inflammatory biomarkers from 0.1pg/ml to 1ug/ml. Results: We demonstrated that the digital nanoneedles achieve the single molecule sensitivity. Therefore, at ultra-low concentrations when antigens that are captured by the nanoneedles follow Poisson statistics, the number of antigens can be quantitated by counting the presence or absence of color changes of individual nanoneedles in a binary fashion. As the protein concentrations increase, the binding events increase accordingly and achieve saturation when all nanoneedles capture more than one protein. Above the digital saturation concentration, an adjacent section of analog nanoneedles perform quantitative analysis based on the level of color change, thus providing a wider dynamic range up to 1ug/ml. Each single analyte area, including both digital and analog sensors, is less than 500um. Therefore, high level multiplex can be achieved by duplicating the detection sensor in a microarray format without loss of sensitivity and dynamic range. Conclusions: The CMOS-compatible NanoMosaic technology provides the cost-effectiveness, sensitivity, dynamic range and multiplexing capacity required to fully integrate patient immune response into therapeutic development and decision making.

21-plex DiLeu Isobaric Tags for High-Throughput Quantitative Proteomics.

Isobaric tags enable multiplexed quantitative analysis of many biological samples in a single LC-MS/MS experiment. As a cost-effective alternative to expensive commercial isobaric tagging reagents, we developed our own custom N,N-dimethylleucine "DiLeu" isobaric tags for quantitative proteomics. Here, we present a new generation of DiLeu tags that achieves 21-plex quantification in high-resolution HCD MS/MS spectra via distinct reporter ions that differ in mass from each other by a minimum of 3 mDa. The 21-plex set retains the compact tag structure and existing isotopologues of the 12-plex set but includes nine new reporter variants formulated with unique configurations of 13C, 15N, and 2H stable isotopes, each synthesized in-house via a stepwise N-monomethylation synthesis strategy using readily available reagents. Thus, multiplexing capacity is expanded significantly, while preserving the performance and low cost of the previous implementation. We show that 21-plex DiLeu tags generate strong reporter ions following HCD fragmentation of labeled peptides acquired on Orbitrap platforms at a minimum of 60,000 resolving power (at 400 m/z), and we demonstrate accurate 21-plex quantification of labeled K562 human cell line protein digests via single-shot nanoLC-MS/MS analysis on a Q Exactive HF system.

Dotdotdot: an automated approach to quantify multiplex single molecule fluorescent in situ hybridization (smFISH) images in complex tissues.

Multiplex single-molecule fluorescent in situ hybridization (smFISH) is a powerful method for validating RNA sequencing and emerging spatial transcriptomic data, but quantification remains a computational challenge. We present a framework for generating and analyzing smFISH data in complex tissues while overcoming autofluorescence and increasing multiplexing capacity. We developed dotdotdot (https://github.com/LieberInstitute/dotdotdot) as a corresponding software package to quantify RNA transcripts in single nuclei and perform differential expression analysis. We first demonstrate robustness of our platform in single mouse neurons by quantifying differential expression of activity-regulated genes. We then quantify spatial gene expression in human dorsolateral prefrontal cortex (DLPFC) using spectral imaging and dotdotdot to mask lipofuscin autofluorescence. We lastly apply machine learning to predict cell types and perform downstream cell type-specific expression analysis. In summary, we provide experimental workflows, imaging acquisition and analytic strategies for quantification and biological interpretation of smFISH data in complex tissues.

Stable Isotope-Triggered Offset Fragmentation Allows Massively Multiplexed Target Profiling on Quadrupole-Orbitrap Mass Spectrometers

Parallel-reaction monitoring (PRM) using high resolution, accurate mass (HR/AM) analysis on quadrupole-Orbitrap mass spectrometers, like the Q Exactive, is one of the most promising approaches for targeted protein analysis. However, PRM has a limited multiplexing capacity, which depends heavily on the reproducibility of peptide retention times. To overcome these limitations, we aimed to establish an easily applicable data acquisition mode that allows retention-time-independent massive multiplexing on Q Exactive mass spectrometers. The presented method is based on data-dependent acquisition and is called pseudo-PRM. In principle, high-intensity stable isotope-labeled peptides are used to trigger the repeated fragmentation of the corresponding light peptides. In this way, pseudo-PRM data can be analyzed like normal PRM data. We tested pseudo-PRM for the target detection from yeast, human cells, and serum, showing good reproducibility and sensitivities comparable to normal PRM. We demonstrated further that pseudo-PRM can be used for accurate and precise quantification of target peptides, using both precursor and fragment ion areas. Moreover, we showed multiplexing of more than 1000 targets in a single run. Finally, we applied pseudo-PRM to quantify vaccinia virus proteins during infection, verifying that pseudo-PRM presents an alternative method for multiplexed target profiling on Q Exactive mass spectrometers.

Metal-Assisted Protein Quantitation (MAPq): Multiplex Analysis of Protein Expression Using Lanthanide-Modified Antibodies with Detection by Inductively Coupled Plasma Mass Spectrometry.

Understanding the complex relationships between genomics, transcriptomics, and proteomics requires the development of more sensitive and rapid methods of multiplexed protein analysis. This is necessary to understand the relationship between cellular responses to environmental stresses, disease progression, and/or drug treatment; however, most methods are limited by low sensitivity, nonspecificity, and minimal multiplexing capacity. To more fully explore the relationship between multiple cellular pathways, we have developed a novel antibody-based multiplex assay using inductively coupled plasma mass spectrometry (ICP-MS), which we term metal-assisted protein quantitation (MAPq). MAPq utilizes lanthanide-conjugated antibodies to simultaneously quantify up to 35 proteins with low pg/mL sensitivity. This method is especially advantageous for low-abundance proteins, a significant limitation of many multiplex MS methods. We observed a limit of detection of 0.5 pg/mL and a limit of quantitation of 5 pg/mL with virtually no background signal. We applied this method to both cultured cells and mouse tissues to investigate changes in low-abundance nuclear and cytoplasmic proteins following drug or environmental stresses. MAPq was found to be at least 10 times more sensitive than Western blots and could detect quantitative changes in protein expression not readily observed using conventional approaches.

Selective Maleylation-Directed Isobaric Peptide Termini Labeling for Accurate Proteome Quantification.

Isobaric peptide termini labeling (IPTL) is an attractive protein quantification method because it provides more accurate and reliable quantification information than traditional isobaric labeling methods (e.g., TMT and iTRAQ) by making use of the entire fragment-ion series instead of only a single reporter ion. The multiplexing capacity of published IPTL implementations is, however, limited to three. Here, we present a selective maleylation-directed isobaric peptide termini labeling (SMD-IPTL) approach for quantitative proteomics of LysC protein digestion. SMD-IPTL extends the multiplexing capacity to 4-plex with the potential for higher levels of multiplexing using commercially available 13C/15N labeled amino acids. SMD-IPTL is achieved in a one-pot reaction in three consecutive steps: (1) selective maleylation at the N-terminus; (2) labeling at the ε-NH2 group of the C-terminal Lys with isotopically labeled acetyl-alanine; (3) thiol Michael addition of an isotopically labeled acetyl-cysteine at the maleylated N-terminus. The isobarically labeled peptides are fragmented into sets of b- and y-ion clusters upon LC-MS/MS, which convey not only sequence information but also quantitative information for every labeling channel and avoid the issue of ratio distortion observed with reporter-ion-based approaches. We demonstrate the SMD-IPTL approach with a 4-plex labeled sample of bovine serum albumin (BSA) and yeast lysates mixed at different ratios. With the use of SMD-IPTL for labeling and a narrow precursor isolation window of 0.8 Th with an offset of −0.2 Th, accurate ratios were measured across a 10-fold mixing range of BSA in a background of yeast proteome. With the yeast proteins mixed at ratios of 1:5:1:5, BSA was detected at ratios of 0.94:2.46:4.70:9.92 when spiked at 1:2:5:10 ratios with an average standard deviation of peptide ratios of 0.34.

Bait Correlation Improves Interactor Identification by Tandem Mass Tag-Affinity Purification-Mass Spectrometry.

The quantitative multiplexing capacity of isobaric tandem mass tags (TMT) has increased the throughput of affinity purification mass spectrometry (AP-MS) to characterize protein interaction networks of immunoprecipitated bait proteins. However, variable bait levels between replicates can convolute interactor identification. We compared the Student's t-test and Pearson's R correlation as methods to generate t-statistics and assessed the significance of interactors following TMT-AP-MS. Using a simple linear model of protein recovery in immunoprecipitates to simulate reporter ion ratio distributions, we found that correlation-derived t-statistics protect against bait variance while robustly controlling type I errors (false positives). We experimentally determined the performance of these two approaches for determining t-statistics under two experimental conditions: irreversible prey association to the Hsp40 mutant DNAJB8H31Q followed by stringent washing, and reversible association to 14-3-3ζ with gentle washing. Correlation-derived t-statistics performed at least as well as Student's t-statistics for each sample and with substantial improvement in performance for experiments with high bait-level variance. Deliberately varying bait levels over a large range fails to improve selectivity but does increase the robustness between runs. The use of correlation-derived t-statistics should improve identification of interactors using TMT-AP-MS. Data are available via ProteomeXchange with identifier PXD016613.

Compact optical asymmetric cryptosystem based on unequal modulus decomposition of multiple color images

In this paper, a silhouette-free and crosstalk-free multiple-color-image cryptosystem is presented based on unequal modulus decomposition (UMD) in the gyrator transform (GT) domains. To the best of our knowledge, it is the first time that the use of cascaded GT, UMD, and inverse-GT operations is demonstrated which features four-orders-of-magnitude higher sensitivity relative to the known GT-UMD cryptosystems. This significantly enlarges its key space. It is demonstrated that this method eliminates the silhouette and cross-talk problems which are well-known in previous interference-based and multiple-image cryptosystems. The polarization and time degrees of freedom (DoFs) are harnessed to realize the proposed cryptosystem using a compact optical system. Also for more compactness, the inverse-GT operation is performed by propagating the beam carrying the image in the reverse direction through the optical GT system. The cryptosystem is verified robust against occlusion, noise, statistical attacks, as well as the chosen-plaintext attack. The quality of the output images is found unaffected by the number of encrypted images which offers ultimately high multiplexing capacity.

Antibody-Free Multiplex Measurement of 23 Human Cytokines in Primary Cell Culture Secretome Using Targeted Mass Spectrometry.

Cytokines are commonly measured by immunoassays; however, these have limited multiplexing capacity, are costly, and can exhibit cross-reactivity. Multiple reaction monitoring (MRM) mass spectrometry is a robust method to quantify analytes with high specificity and multiplexing ability, hence we aimed to investigate its suitability as an alternative cost-effective method for cytokine measurement. Human keratinocyte conditioned media spiked with recombinant cytokines was used as an experimental system to evaluate sensitivity, linearity, and reproducibility of an MRM assay targeting 79 peptides representing 23 human cytokines. Our MRM method was able to identify 21 cytokines by two or more unique peptides and two cytokines by a single unique peptide. In a serum-free matrix, the median LOD and LOQ for cytokine peptides was 130 and 433 pg/mL, respectively. The presence of serum increased median LOD and LOQ by about 2.3-fold. The assay shows excellent replicate consistency with 8% intra- and 12% interday coefficient of variations. We found high pH reversed-phase fractionation a useful tool to increase assay sensitivity with the drawback of increasing its variability by approximately 10%. Overall, our results suggest utility of a multiplex cytokine MRM for routine measurement of secreted cytokines in cellular experiments under low serum conditions. Additional enrichment steps will be required in high complexity matrices such as serum.

Enhancement of the Multiplexing Capacity and Measurement Accuracy of FBG Sensor System Using IWDM Technique and Deep Learning Algorithm

In this article, we are the first to propose deep learning algorithms for intensity wavelength division multiplexing (IWDM)-based self-healing fiber Bragg grating (FBG) sensor network. A deep learning algorithm is proposed to improve the accuracy of measuring the sensing signal of the sensor system. Furthermore, to increase the total number of FBG sensors multiplexed in the sensor network for multipoint measurements, a multiplexing technique called IWDM is proposed. The proposed IWDM-based ring structure FBG sensor network can also have a self-healing purpose to improve the sensor system's reliability and survivability. However, IWDM has unmeasurable gap or crosstalk problems when the number of FBG sensors increases, which causes high sensing signal measurement errors. To solve this problem, a gated recurrent unit (GRU) deep learning algorithm is proposed and experimentally demonstrated. To prove the sensing signal measurement performance of our proposed algorithm, we test the well-trained GRU model using two cases. The first case is when the spectra of FBGs are overlapped as well as the minimum intensity difference between FBGs is 10%, and the second case is when the spectra of FBGs are overlapped as well as the minimum intensity difference between FBGs is 3% which is a very small intensity difference. From the experimental results, the well-trained GRU algorithm achieves high strain sensing signal measurement performance in both cases compared to other algorithms. Therefore, the proposed IWDM based FBG sensor system using deep learning algorithm enhances the multiplexing capacity and survivability of the sensor system, reduces the computational time, and improves strain sensing signal measurement accuracy of FBGs even when FBGs has very small intensity difference and overlap problem.

A Multichannel Spatial-Domain Fiber Cavity Ringdown Pressure Sensor

We propose and demonstrate a multichannel spatial-domain fiber cavity ringdown (FCRD) pressure sensing scheme based on frequency-shifted interferometry (FSI). In contrast to existing multichannel FCRD techniques, multichannel FSI-FCRD measures intensity decay rates of continuous-wave (CW) light from different fiber ringdown cavities (RDCs) in the spatial domain, rather than those of pulse light in the time domain. It shares one CW light source, one slow detector and one slow data collector, which greatly reduces the system cost. We experimentally investigated a dual-channel FSI-FCRD pressure sensing system. The locations and pressures applied of the two FCRDs were obtained by measuring the corresponding ringdown distances. The measurement sensitivities were 0.030 (km $^{-1}\cdot $ MPa−1) and 0.042 (km $^{-1}\cdot $ MPa−1), with the minimum detectable pressure of 0.126 MPa and 0.403 MPa, respectively. By power budge analysis, the maximum sensor number was predicted to be 37 over a 50-km distance under the same experimental settings. The experimental and simulated results show that the proposed scheme has the advantages of low cost, high sensitivity, good linear response and good stability, which can enhance the multiplexing capacity and meet the requirements for multipoint measurement.

Method to improve the tunable capacity of time-resolved encoding to a xanthene dye.

Conventional organic dye encoding is limited by photo-bleaching and spectral overlap, thus restricting the number of distinguishable codes that can be used in practice. The utility of a single dye for increasing additional encoding capacity has yet to be explored. To this end, we firstly report a facile, flexible and green sustainable method to maximize the time-resolved encoding capacity of the xanthene red dye, eosin. By simply adjusting the concentration, pH and viscosity of the eosin solution, eleven distinguishable populations of fluorescence lifetimes were obtained with a short lifetime range of 1.0-3.4ns, which in turn could increase the difficulty of duplication and provide extra high-level security protection. The results provide a facile strategy to increase the temporal multiplexing capacity, and may result in the reuse of existing organic dyes as lifetime-coded polymer microspheres in the fields of information security.