Platform For Protein Detection Research Articles

Ultrasensitive Surface-Enhanced Raman Scattering Platform for Protein Detection via Active Delivery to Nanogaps as a Hotspot.

Surface-enhanced Raman scattering (SERS) is an attractive technique in molecular detection with high sensitivity and label-free characteristics. However, its use in protein detection is limited by the large volume of proteins, hindering its approach to the narrow spaces of hotspots. In this study, we fabricated a Au nanoTriangle plate Array on Gel (AuTAG) as an SERS substrate by attaching a Au nanoTriangle plate (AuNT) arrangement on a thermoresponsive hydrogel surface. The AuTAG acts as an actively tunable plasmonic device, on which the interparticle distance is altered by controlling temperature via changes in hydrogel volume. Further, we designed a Gel Filter Trapping (GFT) method as an active protein delivery strategy based on the characteristics of hydrogels, which can absorb water and separate biopolymers through their three-dimensional (3D) polymer networks. On the AuTAGs, fabricated with AuNTs modified with charged surface ligands to prevent the nonspecific adsorption of analytes to particles, the GFT method helped the delivery of proteins to hotspot areas on the AuNT arrangement. This combination of a AuTAG substrate and the GFT method enables ultrahigh sensitivity for protein detection by SERS up to a single-molecule level as well as a wide quantification concentration range of 6 orders due to their geometric advantages.

A modular cell-free protein biosensor platform using split T7 RNA polymerase.

Conventional laboratory protein detection techniques are not suitable for point-of-care (POC) use because they require expensive equipment and laborious protocols, and existing POC assays suffer from long development timescales. Here, we describe a modular cell-free biosensing platform for generalizable protein detection that we call TLISA (T7 RNA polymerase-Linked ImmunoSensing Assay), designed for extreme flexibility and equipment-free use. TLISA uses a split T7 RNA polymerase fused to affinity domains against a protein. The target antigen drives polymerase reassembly, inducing reporter expression. We characterize the platform, then demonstrate its modularity by using 16 affinity domains against four different antigens with minimal protocol optimization. We show TLISA is suitable for POC use by sensing human biomarkers in serum and saliva with a colorimetric readout within one hour and by demonstrating functionality after lyophilization. Altogether, this technology could have potentially revolutionary impacts, enabling truly rapid, reconfigurable, equipment-free detection of virtually any protein.

Effect of nanoporous membranes thickness in electrochemical biosensing performance: application for the detection of a wound infection biomarker.

Introduction: Nanoporous alumina membranes present a honeycomb-like structure characterized by two main parameters involved in their performance in electrochemical immunosening: pore diameter and pore thickness. Although this first one has been deeply studied, the effect of pore thickness in electrochemical-based nanopore immunosensors has been less taken into consideration. Methods: In this work, the influence of the thickness of nanoporous membranes in the steric blockage is studied for the first time, through the formation of an immunocomplex in their inner walls. Finally, the optimal nanoporous membranes were applied to the detection of catalase, an enzyme related with chronic wound infection and healing. Results: Nanoporous alumina membranes with a fixed pore diameter (60 nm) and variable pore thicknesses (40, 60, 100 μm) have been constructed and evaluated as immunosensing platform for protein detection. Our results show that membranes with a thickness of 40 μm provide a higher sensitivity and lower limit-of-detection (LOD) compared to thicker membranes. This performance is even improved when compared to commercial membranes (with 20 nm pore diameter and 60 μm pore thickness), when applied for human IgG as model analyte. A label-free immunosensor using a monoclonal antibody against anti-catalase was also constructed, allowing the detection of catalase in the range of 50-500 ng/mL and with a LOD of 1.5 ng/mL. The viability of the constructed sensor in real samples was also tested by spiking artificial wound infection solutions, providing recovery values of 110% and 118%. Discussion: The results obtained in this work evidence the key relevance of the nanochannel thickness in the biosensing performance. Such findings will illuminate nanoporous membrane biosensing research, considering thickness as a relevant parameter in electrochemical-based nanoporous membrane sensors.

Alzheimer Plasma Biomarkers Distinguish SuperAgers from their Cognitively Average Peers

AbstractBackgroundSuperAgers are individuals ≥80 years old who were selected for having episodic memory performance that is at least as good as individuals 20‐30 years their junior. We have previously shown that on average SuperAgers show lower neurofibrillary tangle density in the entorhinal cortex than cognitively average controls, which may be one biological factor contributing to superior memory beyond the 8th decade. This preliminary study investigated whether plasma concentrations of Alzheimer and related neurodegenerative biomarkers differ between SuperAgers and their cognitively average peers.MethodSuperAgers (N = 61) and Cognitively Average Controls (N = 28) were enrolled through the SuperAging Research Program. The plasma biomarkers amyloid β peptide 40 (Aβ40), amyloid β peptide 42 (Aβ42), neurofilament light (NFL), phosphorylated tau‐181 (pTau‐181), and glial fibrillary acidic protein (GFAP) were measured in SuperAgers and Cognitively Average participants using the Quanterix single molecule protein detection (SIMOA) platform. Independent samples t‐tests were used for statistical analyses (significance was set at p<0.05, uncorrected).ResultThere were no significant differences in mean age between SuperAgers and Cognitively Average Controls. Concentrations of Aβ42, Aβ40, the ratio of Aβ42/Aβ40, NFL or GFAP did not significantly differ between groups. On average, SuperAgers and Controls showed pTau‐181 levels that were <16.5 pg/mL, falling within the “Unlikely Alzheimer’s” designation according to Quanterix guidelines. SuperAgers showed significantly lower pTau‐181 levels compared to Cognitively Average Controls [M = 5.42 pg/mL (SD = 2.60) vs M = 7.10 pg/mL (SD = 3.14), respectively].ConclusionPlasma phosphotau biomarkers are promising for the assessment of brain integrity versus degeneration across the age‐Alzheimer continuum. The current results provide preliminary support for the contention that the SuperAger brain may be even more removed from the Alzheimer side of the continuum than neurotypical controls.Emily Rogalski and Changiz Geula contributed equally to this study.

Prokaryotic Argonaute Proteins: A New Frontier in Point-of-Care Viral Diagnostics.

The recent pandemic of SARS-CoV-2 has underscored the critical need for rapid and precise viral detection technologies. Point-of-care (POC) technologies, which offer immediate and accurate testing at or near the site of patient care, have become a cornerstone of modern medicine. Prokaryotic Argonaute proteins (pAgo), proficient in recognizing target RNA or DNA with complementary sequences, have emerged as potential game-changers. pAgo present several advantages over the currently popular CRISPR/Cas systems-based POC diagnostics, including the absence of a PAM sequence requirement, the use of shorter nucleic acid molecules as guides, and a smaller protein size. This review provides a comprehensive overview of pAgo protein detection platforms and critically assesses their potential in the field of viral POC diagnostics. The objective is to catalyze further research and innovation in pAgo nucleic acid detection and diagnostics, ultimately facilitating the creation of enhanced diagnostic tools for clinic viral infections in POC settings.

Efficient Three-Dimensional DNA Nanomachine Guided by a Robust Tetrahedral DNA Nanoarray Structure for the Rapid and Ultrasensitive Electrochemical Detection of Matrix Metalloproteinase 2.

Herein, a giant-sized DNA nanoarray was subtly assembled by two kinds of independent tetrahedral DNA structures as the DNA track for a multi-armed three-dimensional (3D) DNA nanomachine to perform signal transduction and amplification efficiently, which was developed as an electrochemical biosensor for the rapid and ultrasensitive detection of matrix metalloproteinase 2 (MMP-2). Impressively, in contrast to conventional DNA walkers with inefficiency, which walked on random DNA tracks composed of a two-dimensional (2D) probe or a one-dimensional (1D) single-stranded (ss)DNA probe, the multi-armed 3D DNA nanomachine from exonuclease III (Exo III) enzyme-assisted target recycling amplification would be endowed with faster reaction speed and better walking efficiency because of the excellent rigidity and orderliness of the tetrahedral DNA nanoarray structure. Once the hairpin H3-label with the signal substance ferrocene (Fc) was added to the modified electrode surface, the multi-armed 3D DNA nanomachine would be driven to move along the well-designed nanoarray tracks by toehold-mediated DNA strand displacement, resulting in most of the ferrocene (Fc) binding to the electrode surface and a remarkable increase in electrochemical signals within 60 min. As a proof of concept, the prepared biosensor attained a low detection limit of 11.4 fg/mL for the sensitive detection of the target MMP-2 and was applied in Hela and MCF-7 cancer cell lysates. As a result, this strategy provided a high-performance sensing platform for protein detection in tumor diagnosis.

Regulation of probe density on upconversion nanoparticles enabling high-performance lateral flow assays

Upconversion nanoparticles (UCNPs)-based fluorescence probes have shown great potential in point-of-care testing (POCT) applications, due to UCNPs’ features of high photostability and background-free fluorescence. Ceaseless improvements of UCNPs-probes have been carried out to increase detection sensitivity and to broaden detection range of UCNPs-based POCT. In this paper, we optimized UCNPs-probes by regulating probe density. The optimization was verified by a traditional lateral flow assay (LFA) platform for C-reactive protein (CRP) detection. Further, the optimized UCNPs-LFA integrating with a home-made benchtop fluorescence analyzer holds the capability to achieve high-performance POCT. Finally, nearly a 20 times sensitivity enhancement with a limit of detection of 0.046 ng/mL and a broad detection range of 0.2–300 ng/mL for CRP detection was obtained. Moreover, the optimized UCNPs-LFA was applied to detecting CRP in clinical serum samples and the detection results were consistent with the clinical test, validating its clinical practicability. The proposed optimization method is also expected to optimize other nanoparticles-based bio-probes for wider POCT application.

An Engineered OmpG Nanopore with Displayed Peptide Motifs for Single‐Molecule Multiplex Protein Detection

AbstractMolecular detection via nanopore, achieved by monitoring changes in ionic current arising from analyte interaction with the sensor pore, is a promising technology for multiplex sensing development. Outer Membrane Protein G (OmpG), a monomeric porin possessing seven functionalizable loops, has been reported as an effective sensing platform for selective protein detection. Using flow cytometry to screen unfavorable constructs, we identified two OmpG nanopores with unique peptide motifs displayed in either loop 3 or 6, which also exhibited distinct analyte signals in single‐channel current recordings. We exploited these motif‐displaying loops concurrently to facilitate single‐molecule multiplex protein detection in a mixture. We additionally report a strategy to increase sensor sensitivity via avidity motif display. These sensing schemes may be expanded to more sophisticated designs utilizing additional loops to increase multiplicity and sensitivity.

An Engineered OmpG Nanopore with Displayed Peptide Motifs for Single-Molecule Multiplex Protein Detection.

Molecular detection via nanopore, achieved by monitoring changes in ionic current arising from analyte interaction with the sensor pore, is a promising technology for multiplex sensing development. Outer Membrane Protein G (OmpG), a monomeric porin possessing seven functionalizable loops, has been reported as an effective sensing platform for selective protein detection. Using flow cytometry to screen unfavorable constructs, we identified two OmpG nanopores with unique peptide motifs displayed in either loop 3 or 6, which also exhibited distinct analyte signals in single-channel current recordings. We exploited these motif-displaying loops concurrently to facilitate single-molecule multiplex protein detection in a mixture. We additionally report a strategy to increase sensor sensitivity via avidity motif display. These sensing schemes may be expanded to more sophisticated designs utilizing additional loops to increase multiplicity and sensitivity.

An aptamer-assisted nanopore strategy with a salt gradient for direct protein sensing.

Nanopore sensing is at the forefront of the technological revolution of the protein research field and has been widely used in molecular diagnosis and molecular dynamics, as well as for various sequencing applications. However, direct protein sensing with biological nanopores is still challenging owing to the large molecular size. Here, we propose an aptamer-assisted nanopore strategy for direct protein sensing and demonstrate its proof-of-concept utilities by experiments with SARS-Cov-2 nucleocapsid protein (NP), the most abundantly expressed viral protein, that is widely used in clinical diagnosis for COVID-19. NP binds with an oligonucleotide-tailed aptamer to form a protein-DNA complex which induces a discriminative two-level pattern of current blockades. We reveal the potential molecular interaction mechanism for the characteristic blockades and identify the salt gradient condition as the dominant factor of the phenomenon. Furthermore, we achieve a high sensitivity of 10 pM for NP detection within one hour and make a preliminary exploration on clinical diagnosis. This work promises a new platform for rapid and label-free protein detection.

Investigation of the enhanced photoactivity of CdS/Bi2MoO6/MoSe2 and its application in antibody-free enzyme-assisted photoelectrochemical strategy for detection of N6-methyladenosine and FTO protein

N6-methyladenosine (m6A) is the most abundant and dynamic reversible modification of mRNA in eukaryotes discovered in the mid-1970s, which is widely involved in various physiological processes. Fat mass and obesity-associated protein (FTO protein) is not only a demethylase for m6A, but also a carcinogenic factor for many human cancers. Therefore, it is very important to build a simple and sensitive platform for simultaneous detection of m6A and FTO protein. To achieve this aim, a novel photoelectrochemical biosensor was constructed with the antibody-free detection strategy of DTT mediated –SH functionalization of m6A triggered by FTO protein catalyzed m6A oxidation. To achieve high detection sensitivity, CdS/Bi2MoO6/MoSe2 system with matched energy gap was employed as photoactive material. To achieve high detection selectivity, the –CH3 of m6A in the captured RNA sequence (m6A RNA) on the electrode surface was oxidized by FTO protein to form hydroxymethyl, followed with the covalent reaction of DTT with hydroxymethyl, making the labeling of m6A with –SH. Finally, a CdS nanoparticle was modified on the electrode through the specific covalent reaction between –SH and Cd, achieving the improvement of the photocurrent with the matched energy band. Under the optimal experimental conditions, the biosensor showed linear range from 0.001 to 50 nM and 0.0005–500 μg/L with the detection limit of 0.37 pM and 0.034 ng/L (S/N = 3) for m6A RNA and FTO protein, respectively. The applicability of the developed method was assessed by investigating the effect of entacapone on FTO protein activity.

Platinum nanoparticles (PtNPs)-based CRISPR/Cas12a platform for detection of nucleic acid and protein in clinical samples

Early rapid screening diagnostic assay is essential for the identification, prevention, and evaluation of many contagious or refractory diseases. The optical density transducer created by platinum nanoparticles (PtNPs) (OD-CRISPR) is reported in the present research as a cheap and easy-to-execute CRISPR/Cas12a-based diagnostic platform. The OD-CRISPR uses PtNPs, with ultra-high peroxidase-mimicking activity, to increase the detection sensitivity, thereby enabling the reduction of detection time and cost. The OD-CRISPR can be utilized to identify nucleic acid or protein biomarkers within an incubation time of 30–40min in clinical specimens. In the case of taking severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) N gene as an instance, when compared to a quantitative reverse transcription-polymerase chain reaction (RT-qPCR), the OD-CRISPR test attains a sensitivity of 79.17% and a specificity of 100%. In terms of detecting prostate-specific antigen (PSA), aptamer-based OD-CRISPR assay achieves the least discoverable concentration of 0.01 ng mL−1. In general, the OD-CRISPR can detect nucleic acid and protein biomarkers, and is a potential strategy for early rapid screening diagnostic tools.

Photoelectrochemical and visual dual-mode sensor for efficient detection of Cry1Ab protein based on the proximity hybridization driven specific desorption of multifunctional probe

Currently, the development of sensitive and visual strategy for Cry1Ab detection, particularly using a switchable dual-mode detection system based on a single component, remains a great challenge. Here, a photoelectrochemical (PEC) and visual dual-mode sensor was designed for Cry1Ab detection based on a proximity hybridization driven multifunctional probe. In the presence of Cry1Ab, specific desorption of the antibody-DNA conjugate was achieved via sufficient proximity hybridization, leading to the selective release of the multifunctional signal probe, i.e., antibody-labeled single-stranded DNA-gold nanoparticles (Ab1-S1-AuNPs). The released Ab1-S1-AuNPs reduced the photocurrent signal and produced a colored response, thereby achieving PEC and visual dual-mode detection based on a single component. Owing to the different signal generation mechanisms, two independent signals were obtained simultaneously, which provided self-verification to improve reliability and accuracy. Taking advantage of the PEC sensitive detection and visual prediction, the dual-mode sensor achieved efficient detection of the Cry1Ab protein. The developed sensor was successfully used to determine Cry1Ab in corn, wheat, and soil samples with satisfactory results. This method offers a promising biosensing platform for the on-site detection of Cry1Ab protein.

A novel label-free universal biosensing platform based on CRISPR/Cas12a for biomarker detection

The development of a biosensing platform with high sensitivity, high specificity, and low cost for the detection of biomarkers, especially one that is programmable and universal, is critical for disease surveillance and diagnosis, yet it remains a difficulty. Herein, we combined the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system with a fluorescent label-free biosensor platform for sensitive and specific detection of disease-related protein, small molecule and nucleic acid. In this strategy, we designed an exonuclease III-mediated target cycle and released a universal trigger chain to stimulate the enzyme activity of CRISPR/Cas12a for additional signal amplification. The hydrolysis of ssDNA-templated silver nanoclusters (ssDNA-Ag NCs) as the reporter probe resulted in a significant decrease of fluorescence intensity. This biosensing platform can be flexibly used to the sensitive and specific determination of protein, small molecule, or microRNA in biological samples by simply transforming the target recognized sequences in the DNA hairpin. In this work, a new label-free sensing system used the fluorescent ssDNA-Ag NCs as the signal output does not need to be marked in advance and has no background signal. In addition, the method has the advantages of low cost, simple operation and high speed, and provides an innovative idea for the development of a powerful clinical diagnosis tool.

A Biosensor Platform for Point-of-Care SARS-CoV-2 Screening.

The COVID-19 pandemic remains a constant threat to human health, the economy, and social relations. Scientists around the world are constantly looking for new technological tools to deal with the pandemic. Such tools are the rapid virus detection tests, which are constantly evolving and optimizing. This paper presents a biosensor platform for the rapid detection of spike protein both in laboratory conditions and in swab samples from hospitalized patients. It is a continuation and improvement of our previous work and consists of a microcontroller-based readout circuit, which measures the capacitance change generated in an interdigitated electrode transducer by the presence either of sole spike protein or the presence of SARS-CoV-2 particles in swab samples. The circuit efficiency is calibrated by its correlation with the capacitance measurement of an LCR (inductance (L), capacitance (C), and resistance (R)) meter. The test result is made available in less than 2 min through the microcontroller’s LCD (liquid-crystal display) screen, whereas at the same time, the collected data are sent wirelessly to a mobile application interface. The novelty of this research lies in the potential it offers for continuous and effective screening of SARS-CoV-2 patients, which is facilitated and enhanced, providing big data statistics of COVID-19 in terms of space and time. This device can be used by individuals for SARS-CoV-2 testing at home, by health professionals for patient monitoring, and by public health agencies for monitoring the spatio-temporal spread of the virus.

An aptasensing platform for detection of heat shock protein 70 kDa (HSP70) using a modified gold electrode with lady fern-like gold (LFG) nanostructure

Early diagnosis of diseases depends on accessibility and the ability to detect associated biomarkers. Using aptamers and equipping signal transducers with nanomaterials have facilitated, accelerated, and provided sensitive and selective nanoaptasensors. In this study, the first aptasensor to detect heat shock protein 70 kDa (HSP70) has been developed by applying a modified gold electrode (GE) with the lady fern-like gold (LFG) nanostructure. The nanostructure solution contained HAuCl4, H2SO4, and histamine and was electrochemically synthesized on the surface of the GE with an average size of ∼20 nm. The analysis to find the optimized time for immobilization of aptamer (a single-stranded RNA) as the biorecognition element on the surface of the working electrode was performed using the open-circuit potential (OCP) technique. This aptasensor could detect HSP70 in a linear range from 0.05 to 75 ng mL−1 with the limit of detection (LOD) ∼ 0.02 ng mL−1. In order to find out about the performance of the designed aptasensor, other analytical analyses for knowing about the figure of metrics were shadowed through reproducibility, stability, regeneration, selectivity, accuracy, and precision experiments.

Poly(ethyleneoxide-block-methylmethacrylate) derived recessed nanodisc electrode array as an ultrasensitive electrochemical sensing platform for immunosensor

In this work, we present the synthesis of amphiphilic diblock copolymer poly(ethylene oxide-block-methylmethacrylate) (PEO-b-PMMA) using atom transfer radical polymerization and demonstrate its application for the fabrication of recessed nanodisk-array electrode (RNE) as a platform for protein detection. For the fabrication of thin-film, PEO-b-PMMA was spin-coated on the silicon wafer and the phase separation was achieved by a unique direct immersion annealing technique followed by washing with the water/methanol solution to remove the PEO phase. The microphase separation and comprehensive porosity characterizations were done using AFM and electron microscopy. Later, the thin film was loaded on the surface of the glassy carbon electrode by wedge transfer technique and the RNE property of the mesoporous thin film was confirmed using cyclic voltammetry (CV). For the protein sensing application, the thin film-coated electrode was biotinylated using EDC-NHS chemistry, followed by the blocking of the non-specific sites using bovine serum albumin (BSA). The reduction in the CV current and the increase in the channel resistance in EIS at each stage of fabrication confirmed the successful immobilization. For the streptavidin detection, the RNE displayed an ultra-low detection limit of 0.15 fg/ml with enhanced selectivity which confirms the potential of PEO-b-PMMA based RNE as an ultrasensitive immunosensor platform for any biomarker detection.

A DNA Electrochemical Sensor via Terminal Protection of Small-Molecule-Linked DNA for Highly Sensitive Protein Detection.

The qualitative and quantitative determination of marker protein is of great significance in the life sciences and in medicine. Here, we developed an electrochemical DNA biosensor for protein detection based on DNA self-assembly and the terminal protecting effects of small-molecule-linked DNA. This strategy is demonstrated using the small molecule biotin and its receptor protein streptavidin (SA). We immobilized DNA with a designed structure and sequence on the surface of the gold electrode, and we named it M1-Biotin DNA. M1-Biotin DNA selectively combines with SA to generate M1-Biotin-SA DNA and protects M1-Biotin DNA from digestion by EXO III; therefore, M1-Biotin DNA remains intact on the electrode surface. M1-Biotin-SA DNA was modified with methylene blue (MB); the MB reporter molecule is located near the surface of the gold electrode, which generates a substantial electrochemical signal during the detection of SA. Through this strategy, we can exploit the presence or absence of an electrochemical signal to provide qualitative target protein determination as well as the strength of the electrochemical signal to quantitatively analyze the target protein concentration. This strategy has been proven to be used for the quantitative analysis of the interaction between biotin and streptavidin (SA). Under optimal conditions, the detection limit of the proposed biosensor is as low as 18.8 pM, and the linear range is from 0.5 nM to 5 μM, showing high sensitivity. The detection ability of this DNA biosensor in complex serum samples has also been studied. At the same time, we detected the folate receptor (FR) to confirm that this strategy can be used to detect other proteins. Therefore, this electrochemical DNA biosensor provides a sensitive, low-cost, and fast target protein detection platform, which may provide a reliable and powerful tool for early disease diagnosis.

Flow-Cytometry Evaluation of Cellular Surface FVIII in Blood Cells and Its Correlation with FVIII Activity and Coagulation

FVIII deficiency or inactivity leads to coagulation malfunction known as Hemophilia A (HA). FVIII overactivity on the contrary is commonly linked to a hypercoagulability state. In steady state, FVIII remains quiescent by forming a non-covalent complex with vWF, and upon coagulation cascade activation, thrombin liberates active FVIII (FVIIIa) from vWF. FVIIIa binds to the plasma membrane of the platelets and other blood cells via its C2 domain phospholipids binding motif to exert its coagulative role.Recently, we developed a reliable Flow-Cytometry (FC) platform for FVIII protein detection (Elnaggar M. et al, 2020). The assay is suitable to measure both intracellular and extracellular FVIII. When applied to human blood it showed that by overexpressing FVIII through a lentiviral-vector-based transduction, a high proportion of circulating FVIII is bound on leukocytes membranes. Leukocyte abundance is reported to be highly associated with thrombotic events but with no clear mechanistic explanation. Therefore, we sought to evaluate the percentage of FVIIIa bound on different leukocyte subsets (T cells, B cells, monocytes and granulocytes) in different clinical scenarios, including HA and other abnormal coagulation states and to correlate it to the plasmatic levels of FVIII.The analysis was conducted on 31 pediatric/young-adult subjects (median age 9y), including patients diagnosed with HA (n=8), patients with coagulopathies (von Willebrand Disease, menorrhagia and unexplained bleedings (n=11) and 1 patient with known high levels of FVIII), and patients without coagulopathies/healthy controls (n=11) referred to Hematology/Oncology clinic at Sidra Medicine-Qatar.Fresh blood samples were analyzed with a Sysmex XN1000 Hematologic Counter for complete blood count and with a BD-Symphony FC for the following surface markers: CD3/CD4/CD8/CD14/CD16/CD19/CD33/Live/Dead and for FVIII protein A2 domain (Ab clone GMA8024), after light-chain Zenon-labeling, with proper IgG controls, as described in the publication above. In parallel, plasma FVIII measurements were performed for clinical diagnostic purposes, and results were correlated.Expectedly, HA patients showed the lowest FVIII percentage in plasma (p<0.0001), while leukocytes' surface FVIII was generally low (on average <1%), trended to a lower rate in patients with coagulopathies and HA compared to healthy-controls and was significantly higher in the patient with hypercoagulability (p=0.0007). Surprisingly, among leukocytes' subpopulations, screened on patients with bleeding diathesis and HA subjects vs healthy-controls, only CD8-surface bound FVIIIa was significantly lower compared to healthy-controls (p=0.015 healthy-controls vs HA, and p=0,0093 healthy-controls vs bleeding diathesis). These results raise the possibility of a CD8-cell mediated controlled binding mechanism regulating FVIII availability in case of biological need of the protein, as bleeding.We did not find any significant correlation between leukocyte-surface bound FVIII and plasma FVIII level, and this further indicates that the differences in FVIIIa leukocyte surface binding are not directly proportional to FVIII availability in the circulation, rather actively regulated by a biological still unknown mechanism.While the study is still ongoing and recruiting other subjects with hypercoagulation state, we showed in this preliminary analysis that our in-house FC platform for FVIII protein detection screens in depth FVIII protein adherence to blood cells, may shed light on coagulation novel mechanisms, and potentially serve as a diagnostic and prognostic tool. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 482: Rapid highly multiplexed immunoprofiling of human fixed tissues by Orion imaging

Abstract The dramatic impact of immune checkpoint inhibitors (ICIs) has focused interest in investigating immune-tumor cell interactions to understand mechanisms of ICI sensitivity and resistance, to identify patients that are responsive to specific treatments, and to develop new therapies. Multiplexed tissue imaging is a highly promising approach to immunoprofile tumors; it can assess many cell types and states within the context of preserved tumor architecture. However, the promise of highly multiplexed tissue imaging remains largely unfulfilled by current methods which are not compatible with pathology workflows. Here we present the development and implementation of Orion™ technology that permits whole-slide rapid single-pass imaging of up to 21 markers from formalin fixed paraffin embedded (FFPE) tissues. This method measures spectra for specific fluorophores to optimally sample the emitted light spectrum and distinguish multiple fluorescence excitation and emission channels across the spectral range of optical microscopes (~400-900 nm). To establish the utility of the Orion™ platform for immuno-phenotyping and immune checkpoint protein detection, we created an immunoprofiling panel of 21 qualified antibodies and labelled with fluorophores to subdivide the available emission spectrum in the 438-893 nm range. The panel includes markers that define subsets of T cells and macrophages. FFPE sections of human tonsil and matched primary and brain metastatic lung adenocarcinoma were stained and imaged in a single pass demonstrating staining patterns consistent with known micro-anatomic compartments and cell types in tonsil and identifying immune cell subtypes and heterogeneous checkpoint protein expression in tumor samples. An unexpected benefit of imaging with the Orion platform is the reduction of autofluorescence which is highly advantageous for the detection of proteins, like PD-L1, that function at very low levels. Orion imaging promises to accelerate discovery of predictive and prognostic biomarkers, enable pharmacodynamics study of immuno-oncology drugs undergoing clinical trials and ultimately provide clinically actionable diagnostic tests. Citation Format: Jia-Ren Lin, Daniel E. Campton, Jeremy Cooper, Yu-An Chen, Erin F. McCarty, Keith L. Ligon, Eric P. Kaldjian, Kyla Teplitz, Steve Reese, Sandro Santagata, Peter K. Sorger. Rapid highly multiplexed immunoprofiling of human fixed tissues by Orion imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 482.