Multiplexing Capability Research Articles

Activatable Fluorescence-Encoded Nanoprobes Enable Simple Multiplexed RNA Imaging in Live Cells.

Benefiting from superior programmable performance and flexible design of DNA technologies, a variety of single-molecule RNA fluorescence imaging methodologies have been reported. However, the multiplexing capability is restricted owing to the spectral overlap of fluorophores. To overcome this limitation, some inspiring multiplex imaging strategies have been developed, but in practice, it remains challenging to achieve convenient and rapid imaging in live cells due to complex designs and additional pretreatments to increase cell permeability. Here, we report an activatable fluorescence-encoded nanoprobe (AFENP) strategy, through which fluorescence-encoded functional modules for qualitative analysis and activated nucleic acid assemblies functional modules for quantitative testing enable simple multiplexed RNA imaging in single live cells. As a proof of principle, by two distinguishable fluorophores (fluorescein and rhodamine B) and their seven distinctly differentiated intensity levels, self-assembled AFENP enables simplified and quick simultaneous in situ detection and imaging of seven types of targets in live single cells because the fluorescent quantitative signal is activated only in the presence of target avoiding the washing procedures and additional pretreatment to increase cell permeability is undesired. We expect that this practical single-cell analysis platform will be adopted for multiple gene expression analysis and imaging in live cells on account of its simplicity and multiplex capability.

Controllable Local Propagation Environment to Maximize the Multiplexing Capability of Massive MIMO Systems

The capability of controlling and modifying wireless propagation channels is one of the prerogatives of beyond-5G systems. In this paper, we propose the use of a controllable local propagation environment surrounding the terminals, and analyze its positive effect on the multiplexing capability of massive MIMO systems. In particular, we focus on using a few switched passive elements surrounding each terminal. In this way, the modification of the propagation environment is not realized by means of a single structure, as in reconfigurable intelligent surfaces (RIS), but is achieved by the cooperative work of all the terminals. By employing numerical simulations, we show that the proposed system outperforms its non-reconfigurable counterpart in terms of the number of contemporary connected users. Moreover, the optimized system enables a substantial increase in the minimum received power by the terminals, thus guaranteeing superior channel fairness.

A User-Centric 3D-Printed Modular Peristaltic Pump for Microfluidic Perfusion Applications.

Microfluidic organ-on-a-chip (OoC) technology has enabled studies on dynamic physiological conditions as well as being deployed in drug testing applications. A microfluidic pump is an essential component to perform perfusion cell culture in OoC devices. However, it is challenging to have a single pump that can fulfil both the customization function needed to mimic a myriad of physiological flow rates and profiles found in vivo and multiplexing requirements (i.e., low cost, small footprint) for drug testing operations. The advent of 3D printing technology and open-source programmable electronic controllers presents an opportunity to democratize the fabrication of mini-peristaltic pumps suitable for microfluidic applications at a fraction of the cost of commercial microfluidic pumps. However, existing 3D-printed peristaltic pumps have mainly focused on demonstrating the feasibility of using 3D printing to fabricate the structural components of the pump and neglected user experience and customization capability. Here, we present a user-centric programmable 3D-printed mini-peristaltic pump with a compact design and low manufacturing cost (~USD 175) suitable for perfusion OoC culture applications. The pump consists of a user-friendly, wired electronic module that controls the operation of a peristaltic pump module. The peristaltic pump module comprises an air-sealed stepper motor connected to a 3D-printed peristaltic assembly, which can withstand the high-humidity environment of a cell culture incubator. We demonstrated that this pump allows users to either program the electronic module or use different-sized tubing to deliver a wide range of flow rates and flow profiles. The pump also has multiplexing capability as it can accommodate multiple tubing. The performance and user-friendliness of this low-cost, compact pump can be easily deployed for various OoC applications.

Genomic profiling of NGS-based ctDNA from Chinese non-small cell lung cancer patients.

Cell-free circulating tumor DNA (ctDNA) in plasma enables rapid and repeat testing of actionable mutations. Next-generation sequencing (NGS) is an attractive platform for multiplex sequencing capabilities compared to traditional methods such as PCR. The purpose of this study is to evaluate the value of the NGS-based ctDNA assay and to identify the genomic alteration profile of ctDNA in real-world Chinese non-small cell lung (NSCLC) patients. In total, 294 Chinese patients with pathological diagnosis of Phase III-IV NSCLC were enrolled. 3-4mL peripheral blood was collected and NGS-based analysis was carried out using a 20-gene panel. The analytical sensitivity and specificity of ctDNA NGS-based assay was validated using droplet digital PCR (ddPCR). We have tested 570 sites from 286 samples using ddPCR, which included 108 positive sites and 462 negative sites from NGS results, and the concordance rate was 99.8% (418/419) for single-nucleotide variants (SNVs) and 96.7% (146/151) for insertions and deletions (InDels). The most frequent genes were TP53 (32%), EGFR (31.97%), KRAS (6.46%), PIK3CA (4.76%), and MET (4.08%). Exon 19 deletion (19del) was the most common alteration in EGFR and G12C was the most common alteration in KRAS. Furthermore, the detection rate of TP53 was higher in the male and patients with squamous cell carcinoma. We also found the prevalence of TP53 in L858R was higher than in 19del (61.29% vs. 40%; p = 0.1115). The results indicate that the results of NGS-based ctDNA assay are highly consistent with ddPCR. In Chinese NSCLC patients, TP53 mutation was more frequently associated with male and squamous cell carcinoma. The prevalence of concomitant mutations in L858R may be different from that in 19del.

Isobaric Stable Isotope N‐Phosphorylation Labeling (iSIPL) for Ultrasensitive Proteome Quantification

AbstractStable isotope chemical labeling methods have been widely used for high‐throughput mass spectrometry (MS)‐based quantitative proteomics in biological and clinical applications. However, the existing methods are far from meeting the requirements for high sensitivity detection. In the present study, a novel isobaric stable isotope N‐phosphorylation labeling (iSIPL) strategy was developed for quantitative proteome analysis. The tryptic peptides were selectively labeled with iSIPL tag to generate the novel reporter ions containing phosphoramidate P−N bond with high intensities under lower collision energies. iSIPL strategy are suitable for peptide sequencing and quantitative analysis with high sensitivity and accuracy even for samples of limited quantity. Furthermore, iSIPL coupled with affinity purification and mass spectrometry was applied to measure the dynamics of cyclin dependent kinase 9 (CDK9) interactomes during transactivation of the HIV‐1 provirus. The interaction of CDK9 with PARP13 was found to significantly decrease during Tat‐induced activation of HIV‐1 gene transcription, suggesting the effectiveness of iSIPL strategy in dynamic analysis of protein‐protein interaction in vivo. More than that, the proposed iSIPL strategy would facilitate large‐scale accurate quantitative proteomics by increasing multiplexing capability.

Isobaric Stable Isotope N-Phosphorylation Labeling (iSIPL) for Ultrasensitive Proteome Quantification.

Stable isotope chemical labeling methods have been widely used for high-throughput mass spectrometry (MS)-based quantitative proteomics in biological and clinical applications. However, the existing methods are far from meeting the requirements for high sensitivity detection. In the present study, a novel isobaric stable isotope N-phosphorylation labeling (iSIPL) strategy was developed for quantitative proteome analysis. The tryptic peptides were selectively labeled with iSIPL tag to generate the novel reporter ions containing phosphoramidate P-N bond with high intensities under lower collision energies. iSIPL strategy are suitable for peptide sequencing and quantitative analysis with high sensitivity and accuracy even for samples of limited quantity. Furthermore, iSIPL coupled with affinity purification and mass spectrometry was applied to measure the dynamics of cyclin dependent kinase 9 (CDK9) interactomes during transactivation of the HIV-1 provirus. The interaction of CDK9 with PARP13 was found to significantly decrease during Tat-induced activation of HIV-1 gene transcription, suggesting the effectiveness of iSIPL strategy in dynamic analysis of protein-protein interaction in vivo. More than that, the proposed iSIPL strategy would facilitate large-scale accurate quantitative proteomics by increasing multiplexing capability.

Activatable Raman Probes Utilizing Enzyme-Induced Aggregate Formation for Selective Ex Vivo Imaging

Detecting multiple enzyme activities simultaneously with high spatial specificity is a promising strategy to investigate complex biological phenomena, and Raman imaging would be an excellent tool for this purpose due to its high multiplexing capabilities. We previously developed activatable Raman probes based on 9CN-pyronins, but specific visualization of cells with target enzyme activities proved difficult due to leakage of the hydrolysis products from the target cells after activation. Here, focusing on rhodol bearing a nitrile group at the position of 9 (9CN-rhodol), we established a novel mechanism for Raman signal activation based on a combination of aggregate formation (to increase local dye concentration) and the resonant Raman effect along with the bathochromic shift of the absorption, and utilized it to develop Raman probes. We selected the 9CN-rhodol derivative 9CN-JCR as offering a suitable combination of increased stimulated Raman scattering (SRS) signal intensity and high aggregate-forming ability, resulting in good retention in target cells after probe activation. By using isotope-edited 9CN-JCR-based probes, we could simultaneously detect β-galactosidase, γ-glutamyl transpeptidase, and dipeptidyl peptidase-4 activities in live cultured cells and distinguish cell regions expressing target enzyme activity in Drosophila wing disc and fat body ex vivo.

Multiplex Digital Methylation-Specific PCR for Noninvasive Screening of Lung Cancer.

There remains tremendous interest in developing liquid biopsy assays for detection of cancer-specific alterations, such as mutations and DNA methylation, in cell-free DNA (cfDNA) obtained through noninvasive blood draws. However, liquid biopsy analysis is often challenging due to exceedingly low fractions of circulating tumor DNA (ctDNA), necessitating the use of extended tumor biomarker panels. While multiplexed PCR strategies provide advantages such as higher throughput, their implementation is often hindered by challenges such as primer-dimers and PCR competition. Alternatively, digital PCR (dPCR) approaches generally offer superior performance, but with constrained multiplexing capability. This paper describes development and validation of the first multiplex digital methylation-specific PCR (mdMSP) platform for simultaneous analysis of four methylation biomarkers for liquid-biopsy-based detection of non-small cell lung cancer (NSCLC). mdMSP employs a microfluidic device containing four independent, but identical modules, housing a total of 40 160 nanowells. Analytical validation of the mdMSP platform demonstrates multiplex detection at analytical specificities as low as 0.0005%. The clinical utility of mdMSP is also demonstrated in a cohort of 72 clinical samples of low-volume liquid biopsy specimens from patients with computed tomography(CT)-scan indeterminant pulmonary nodules, exhibiting superior clinical performance when compared to traditional MSP assays for noninvasive detection of early-stage NSCLC.

A DNA copy number alteration classifier as a prognostic tool for prostate cancer patients.

Distinguishing between true indolent and potentially life-threatening prostate cancer is challenging in tumours displaying clinicopathologic features associated with low or intermediate risk of relapse. Several somatic DNA copy number alterations (CNAs) have been identified as potential prognostic biomarkers, but the standard cytogenetic method to assess them has a limited multiplexing capability. Multiplex ligation-dependent probe amplification (MLPA) targeting 14 genes was optimised to survey 448 tumours of patients with low or intermediaterisk (Grade Group 1-3, Gleason score ≤7) who underwent radical prostatectomy. A 6-gene CNA classifier was developed using random survival forest and Cox proportional hazard modelling to predict biochemical recurrence. The classifier score was significantly associated with biochemical recurrence after adjusting for standard clinicopathologic variables and the known prognostic index CAPRA-S score with a hazard ratio of 2.17 and 1.80, respectively (n = 406, P < 0.01). The prognostic value of this classifier was externally validated in published CNA data from three radical prostatectomy cohorts and one radiation therapy pre-treatment biopsy cohort. The 6-gene CNA classifier generated by a single MLPA assay compatible with the small quantities of DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue specimens has the potential to improve the clinical management of patients with low or intermediaterisk disease.

Abstract 2314: Development of a 6-color multiplex Crystal Digital PCR assay for the detection of ESR1 and PIK3CA mutations in the plasma of metastatic breast cancer patients

Abstract Blood-based liquid biopsies have become a real asset for patient treatment management in the field of precision oncology over the past few years, as they allow quick and easy access to tumor genetic alterations of interest. The analysis of circulating tumor DNA (ctDNA) from these liquid biopsies is a particularly well-suited strategy for longitudinal monitoring of patients. In patients with HR+/HER2- metastatic breast cancer (MBC), early detection of ESR1 and PIK3CA mutations is a key element to better personalize disease management. Among the technologies currently used for liquid biopsies, digital PCR (dPCR) has the advantage of being among the fastest and least expensive, providing robust results along with unmatched sensitivity as compared to other methods. Until recently, however, dPCR has been previously limited by multiplex capability. The recent availability of the 6-color version of the naica® platform (Stilla Technologies) has allowed us to develop a single-well multiplexed assay that combines an 11 pathogenic mutation ESR1 assay with a 21 mutation PIK3CA assay, for a combined total of 32 ESR1 and PIK3CA mutations. Here we present data on this new high-plex dPCR panel. As ESR1 and PIK3CA analyses are often requested together, this new screening assay represents a significant saving of time and resources for the analysis of our MBC patients with a relative ease of implementation and analysis. Citation Format: Veronique Quillien, Julien Corne, Thibault De la Motte Rouge, Fanny Le Du, Florence Godey. Development of a 6-color multiplex Crystal Digital PCR assay for the detection of ESR1 and PIK3CA mutations in the plasma of metastatic breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2314.

Abstract 5059: High-throughput monitoring of proteoforms and pathways through multiplexed and customizable mass spectrometry assay panels

Abstract Proteins represent a key class of analytes in pharmacodynamic (PD) analysis, an essential component in preclinical and clinical studies which inform about a biological system’s response to drug treatment. Pharmacodynamic signatures typically comprise target engagement marker and downstream pathway markers. With recent development in targeted therapies and novel therapeutic modalities such as protein degraders, the scope of PD has widened to cover predictive and stratification biomarkers which can inform patient selection and contribute to further understanding of potential drug resistance. Using peptides as surrogate for proteins of interest, mass spectrometry (MS)-based approach alleviates the barriers of reagent specificity of antibody-based methods which can be affected by species and matrices used in preclinical drug development. Moreover, the use of stable-isotope labeled standard (SIS) reference peptides grants targeted MS assays unraveled specificity and the ability to determine absolute quantity of a given analyte. Harnessing the power of targeted MS, we present an oncology-focused assay repository which contains 804 peptides corresponding to 582 proteins. All 804 peptide surrogates were chosen under strict selection criteria such as uniqueness/proteotypicity and stability. Off-the-shelf availability (PQ500 kit) of SIS reference peptides for all 582 proteins facilitates the deployment of any of these assays in a plug-and-play manner. The collection of 582 protein targets cover &amp;gt; 180 cellular pathways (Reactome pathways e.g. cytokine signaling, cell-cell communication, activation of matrix metalloproteinases) together with 49 FDA-approved drug targets. To further evaluate the performance of these assays, we carried out a case control study comprising plasma samples from 5 patient groups (lung, colorectal, pancreatic, breast and prostate cancer) and age and gender-matched healthy subjects. In total, we measured absolute quantities of 582 proteins across 180 plasma samples while validating the expression profiles of well-characterized biomarkers in patient plasma. To conclude, we’ve demonstrated the application of targeted MS approach for the routine analysis of pharmacodynamic biomarkers. Its multiplexing capability, together with the adaptability to various species allow this method to maneuver through preclinical and clinical scenarios. Citation Format: Sebastian Müller, Véronique Laforte, Simonas Savickas, Maik Müller, Yuehan Feng, Lukas Reiter. High-throughput monitoring of proteoforms and pathways through multiplexed and customizable mass spectrometry assay panels. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5059.

Abstract 3588: Multiplex proteomics profiling with the imaging mass cytometry (IMC) identifies immune cell phenotypes associated with high NOS2/COX2 expression in ER- negative and TNBC patients

Abstract Elevated expression of Inducible Nitric oxide Synthase-2 (NOS2) occurs during acute and chronic inflammation and wound healing processes however, it is also a predictor of bad prognosis in aggressive ER-negative tumors. Overexpression of NOS2 correlates with other prognostic biomarkers making it a unique multifunctional oncoprotein. Cyclooxygenase-2 (COX2) is another enzyme associated with cancer inflammation and is overexpressed in &amp;gt;50% of breast cancers. In our previous studies, we have shown that NOS2/COX2 co-expression is a strong predictor of poor clinical outcome and promotes CD8+ T cell exclusion from tumor epithelia in Triple-negative breast cancers (TNBC). This observation was further explored using multiplexing proteomics technologies. Imaging Mass Cytometry (IMC) has revolutionized tumor microenvironment research by allowing the interrogation of over 35 protein markers simultaneously for the elucidation of the spatial distribution of immune and tumor cells at sub-cellular resolution. Herein, we used multiplex capability IMC to explore the interactions of immune and tumor cell counterparts in ER-negative and TNBC tumors. We employed Formalin-fixed paraffin-embedded (FFPE) tissue from 26 ER-negative and TNBC samples from a clinical cohort of African American breast cancer patients in the Baltimore MD area. We developed a panel of 15 protein metal-labeled antibody markers including antibodies targeting NOS2, COX2, T cells (CD45, CD3, CD4, CD8,) endothelium (CD31), monocytes (CD14), macrophages (CD68), basal markers (α-SMA), cancer stem cells (CD44v6), cancer cells (Pan-CK, B7H4, Cadherin), and serine proteases (granzyme B) secreted by T cells or Natural Killer (NK) cells. Single section slides of 4 µm size were stained with the cocktail of metal-labeled antibodies. Multiple regions of interest (ROIs) representing the tumor and immune cells compartments and tumor-facing stromal cell areas were ablated with the Hyperion Imaging system. The data analysis was performed by the Bodenmiller lab pipeline and VisioPharm software packages. Our preliminary data suggest that patient tumors with high or low NOS2/COX2 expression showed variable levels of CD68 positive macrophage population and T cells. We are currently analyzing the remaining data to understand the cell phenotypes associated with the immunosuppressive and immune-rich clinical samples and performing neighborhood analysis of tumor cells to immune cells and vessels. Citation Format: Milind Pore, Lisa Ridnour, Paul Mallory, Ashley Cardamone, Stephen Lockett, William Bocik, David Wink. Multiplex proteomics profiling with the imaging mass cytometry (IMC) identifies immune cell phenotypes associated with high NOS2/COX2 expression in ER- negative and TNBC patients. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3588.

Abstract 2759: Real-time cellular target engagement and protein quantification for drug discovery

Abstract Preclinical assessment of direct drug-target engagement and analysis of binding kinetics within the cellular environment is essential for development of safer and more effective therapeutics. Infusion of such critical data into early drug discovery would significantly reduce the failure rate of new drug candidates, accelerate drug discovery, and validate repurposing of existing drugs. Described here is a novel drug-target engagement technology that can sensitively interrogate direct binding of drugs to cellular, bacterial, or viral protein targets within the physiological environment. Micro-Tag cell target engagement technology is based on complementation of a small 15-amino acid subunit with a large subunit into an active RNA-processing enzyme. The small subunit can be cloned to any drug target for transient or stable expression using existing tools such as CRISPR. Upon complementation, the active enzyme cleaves a FRET-based oligonucleotide substrate resulting in rapid generation of fluorescent signal that can be quantified in real time. The Micro-Tag technology enables in-cell quantitation of drug target levels using qPCR systems. This is the next-generation of target engagement technology that allows for real-time monitoring of drug-target interaction in the cell. We show here data demonstrating direct engagement of several reference compounds and novel small molecules with high-profile cancer targets such as K-RAS, MTH1, EGFR, and UBE2N. Selectivity of these drugs to the targets in the cell is further delineated using their mutant counterparts as well as inactive stereoisomer compounds. The Micro-Tag cell target engagement technology provides the power of cell target engagement to a large family of target proteins. It can be employed for high-throughput screens directed at initial on-target ranking or med-chem optimization of drug candidates. It is amenable for interrogation of drug candidates across various modalities: small molecules, peptides, antibodies and PROTACs. Importantly, this next-generation target engagement technology seamlessly integrates with qPCR systems, fluorescence microscopy, live-cell microscopy, FACS analysis, and offers multiplexing capability, thus allowing for further mechanistic insight. Citation Format: Ivan Babic, Nikolas Bryan, Claire Cunningham, Avery Sampson, Daniel Starczynowski, Elmar Nurmemmedov. Real-time cellular target engagement and protein quantification for drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2759.

Abstract 4636: High-plex co-detection of RNA and protein to explore tumor-immune interactions utilizing RNAscope with Imaging Mass Cytometry

Abstract Purpose The next breakthroughs in immuno-oncology will be driven by high-plex tools that decipher the spatial arrangement of different cell types within the tumor microenvironment (TME). Imaging Mass Cytometry™ (IMC™) is a proven tool for the study of complex cellular interactions in the TME. It utilizes CyTOF® technology for simultaneous assessment of 40-plus protein markers at subcellular resolution without spectral overlap or background autofluorescence, thus providing unprecedented insight into the organization and function of the TME. Despite this, some protein targets are challenging to include in IMC as they have very few or no commercial antibodies available. Moreover, although cellular identity can easily be deciphered through detection of protein targets, knowledge of the cell’s transcriptome improves understanding of cellular function and activation state. Here, we present a robust and reliable workflow that combines the highly sensitive and specific RNAscope™ technology for RNA detection with the multiplexing capability of IMC to visualize key RNA and protein markers in the same tumor samples. Methods The RNAscope HiPlex v2 assay was combined with protein detection on the Hyperion+™ Imaging System to evaluate expression of both RNA and protein targets in formalin-fixed, paraffin-embedded (FFPE) tumor tissue microarray (TMA). The RNAscope assay was used with 12 target RNA marker probes and associated metal-labeled detection probes, suitable for use in IMC. The RNAscope HiPlex v2 assay workflow was followed as recommended up until applying the fluoros. Instead of using the fluoros, metal-conjugated probes were used for RNA detection. Metal-conjugated antibodies were used for protein detection in the same tissue. Results Target protein markers identified a range of immune cells, tumor cells, stromal cells, endothelial cells, and extracellular matrix. Co-detection of RNA and protein allowed visualization of several cytokines and chemokines, such as CXCL13, CXCL9, CXCL10, IFNγ, IL10, IL8, and IL1B, enabling the identification of the cellular source of these secreted factors. Additionally, immune cell subpopulations and their activation states were visualized using marker-specific antibodies. Immune cell hubs associated with anti-tumor immune responses were detected in tumor niches across the TMA. Conclusions Overall, combining RNAscope with protein co-detection on the IMC platform allowed simultaneous visualization of RNA and protein targets to interrogate the tumor microenvironment. The single-cell resolution offered by the RNAscope assay provides superior sensitivity for RNA detection in addition to the existing protein-detection capability of the IMC platform. This workflow can be applied to FFPE tissues to study therapeutic efficacy, stratify patients based on inflammatory signature, and study cell-cell interactions within the TME. Citation Format: James Pemberton, Smriti Kala, Anushka Dikshit, Clinton Hupple. High-plex co-detection of RNA and protein to explore tumor-immune interactions utilizing RNAscope with Imaging Mass Cytometry. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4636.

WEAVE-StePS: A stellar population survey using WEAVE at WHT

Context. The upcoming new generation of optical spectrographs on four-meter-class telescopes will provide valuable opportunities for forthcoming galaxy surveys through their huge multiplexing capabilities, excellent spectral resolution, and unprecedented wavelength coverage. Aims. WEAVE is a new wide-field spectroscopic facility mounted on the 4.2 m William Herschel Telescope in La Palma. WEAVE-StePS is one of the five extragalactic surveys that will use WEAVE during its first five years of operations. It will observe galaxies using WEAVE MOS (∼950 fibres distributed across a field of view of ∼3 square degrees on the sky) in low-resolution mode (R ∼ 5000, spanning the wavelength range 3660 − 9590 Å). Methods. WEAVE-StePS will obtain high-quality spectra (S/N ∼ 10 Å−1 at R ∼ 5000) for a magnitude-limited (IAB = 20.5) sample of ∼25 000 galaxies, the majority selected at z ≥ 0.3. The survey goal is to provide precise spectral measurements in the crucial interval that bridges the gap between LEGA-C and SDSS data. The wide area coverage of ∼25 square degrees will enable us to observe galaxies in a variety of environments. The ancillary data available in each of the observed fields (including X-ray coverage, multi-narrow-band photometry and spectroscopic redshift information) will provide an environmental characterisation for each observed galaxy. Results. This paper presents the science case of WEAVE-StePS, the fields to be observed, the parent catalogues used to define the target sample, and the observing strategy that was chosen after a forecast of the expected performance of the instrument for our typical targets. Conclusions. WEAVE-StePS will go back further in cosmic time than SDSS, extending its reach to encompass more than ∼6 Gyr. This is nearly half of the age of the Universe. The spectral and redshift range covered by WEAVE-StePS will open a new observational window by continuously tracing the evolutionary path of galaxies in the largely unexplored intermediate-redshift range.

Multiplexed photoinjector optimization for high-repetition-rate free-electron lasers

The multiplexing capabilities of superconducting X-ray free-electron lasers (FELs) have gained much attention in recent years. The demanding requirements for photon properties from multiple undulator lines necessitate more flexible beam manipulation techniques to achieve the goal of “beam on demand”. In this paper, we investigate a multiplexed configuration for the photoinjector of high-repetition-rate FELs that aims to simultaneously provide low-emittance electron beams of different charges. A parallel, multi-objective genetic algorithm is implemented for the photoinjector parameter optimization. The proposed configuration could drastically enhance the flexibility of beam manipulation to improve multiplexing capabilities and realize the full potential of the facility.

Label-Free Saliva Test for Rapid Detection of Coronavirus Using Nanosensor-Enabled SERS.

The recent COVID-19 pandemic has highlighted the inadequacies of existing diagnostic techniques and the need for rapid and accurate diagnostic systems. Although molecular tests such as RT-PCR are the gold standard, they cannot be employed as point-of-care testing systems. Hence, a rapid, noninvasive diagnostic technique such as Surface-enhanced Raman scattering (SERS) is a promising analytical technique for rapid molecular or viral diagnosis. Here, we have designed a SERS- based test to rapidly diagnose SARS-CoV-2 from saliva. Physical methods synthesized the nanostructured sensor. It significantly increased the detection specificity and sensitivity by ~ten copies/mL of viral RNA (~femtomolar concentration of nucleic acids). Our technique combines the multiplexing capability of SERS with the sensitivity of novel nanostructures to detect whole virus particles and infection-associated antibodies. We have demonstrated the feasibility of the test with saliva samples from individuals who tested positive for SARS-CoV-2 with a specificity of 95%. The SERS-based test provides a promising breakthrough in detecting potential mutations that may come up with time while also preparing the world to deal with other pandemics in the future with rapid response and very accurate results.

Photothermal Responsive Digital Polymerase Chain Reaction Resolving Exosomal microRNAs Expression in Liver Cancer.

Exosomal microRNAs have been studied as a good source of noninvasive biomarkers due to their functions in genetic exchange between cells and have been already well documented in many biological activities; however, inaccuracy remains a key challenge for liver cancer surveillance. Herein, a versatile duplex photothermal digital polymerase chain reaction (PCR) strategy combined with a lipid nanoparticle-based exosome capture approach is proposed to profile microRNAs expression through a 3-h easy-to-operate process. The microfluidically-generated molybdenum disulfide-nanocomposite-doped gelatin microcarriers display attractive properties as a 2-4°C s-1 ramping-up rate triggered by near-infrared and reversible sol-gel transforming in step with PCR activation. To achieve PCR thermocycling, the corresponding irradiation coordinating with fan cooling are automatically performed via a homemade control module with programs. Thus, taking the multiplexing capability of dual-color labeling, 19-31 folds higher in exosomal microRNA-200b-3p and microRNA-21-5p, and tenfold lower in microRNA-22-3p expressions relative to the control microRNA-26a-5p are quantified in two liver cancer cells (Huh7 and HepG2) than in those from the healthy cells. It is believed that this exosomal microRNA genotyping method would be highly applicable for liver cancer diagnostics.

Two-photon fluorescence imaging using a tunable spectral window based on fiber supercontinuum.

Two-photon excitation fluorescence (TPEF) microscopy has evolved into a versatile tool in biological research. However, the multiplexing capability of TPEF microscopy is limited by the narrow spectral bandwidth of the light source. In this study, we apply a photonic crystal fiber in TPEF microscopy to broaden the excitation source bandwidth. We tuned the spectral window using a spatial light modulator as a programmable diffraction grating that was placed behind a prism pair. In addition, we combined a grating pair to compensate for dispersion to improve the two-photon excitation efficiency. The combination of a broad spectrum and a programmable grating enabled fast spectral window tuning rate on a time scale of tens of milliseconds. We demonstrate the performance of our method by imaging live B16 cells labeled with four emission spectrum overlapped fluorescent proteins.

A multiplex method for detection of SARS-CoV-2 variants based on MALDI-TOF mass spectrometry

The recent outbreak of the coronavirus disease 2019 (COVID-19) pandemic and the continuous evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have highlighted the significance of new detection methods for global monitoring and prevention. Although quantitative reverse transcription PCR (RT-qPCR), the current gold standard for diagnosis, performs excellently in genetic testing, its multiplexing capability is limited because of the signal crosstalk of various fluorophores. Herein, we present a highly efficient platform which combines 17-plex assays with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), enabling the targeting of 14 different mutation sites of the spike gene. Diagnosis using a set of 324 nasopharyngeal swabs or sputum clinical samples with SARS-CoV-2 MS method was identical to that with the RT-qPCR. The detection consistency of mutation sites was 97.9% (47/48) compared to Sanger sequencing without cross-reaction with other respiratory-related pathogens. Therefore, the MS method is highly potent to track and assess SARS-CoV-2 changes in a timely manner, thereby aiding the continuous response to viral variation and prevention of further transmission.