Platform For Protein Detection Research Articles

Nano-immunosorbent assay based on Cas12a/crRNA for ultra-sensitive protein detection

Apart from the great potential in genome editing, the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas system has recently been widely used in biosensing. However, due to the complex and inefficient signal conversion strategies, most of the works focused on nucleic acid analysis rather than protein biomarkers. Herein, by employing DNA-AuNPs (gold nanoparticles) nanotechnology to activate trans-cleavage activity of CRISPR/Cas12a, a universal signal transduction strategy was established between trans-cleavage of CRISPR/Cas12a and protein analytes. As a result, a sensitive platform was developed for sensing carcinoembryonic antigen (CEA) and prostate specific-antigen (PSA) biomarkers, which was designated as Nano-CLISA (Nano-immunosorbent assay based on Cas12a/crRNA). Nano-CLISA was directly employed to test PSA in clinical samples, indicating its great potential in practical detection. This platform has been used to quantitatively analyze protein at attomolar levels, which was 1000-fold more sensitive than traditional ELISA, and the detection range is 15 times wider than that of traditional ELISA.

Immunoaffinity-Based Graphene Field-Effect Transistor Sensor Platform for Fast Detection of S1 Subunit Protein from COVID-19

The epidemic of the novel coronavirus (COVID-19) pneumonia touches the nerve of every person. To prevent and control the epidemic, early diagnosis must be addressed. Nucleic acid polymerase chain reaction (PCR) is currently used worldwide to identify positive carriers of COVID-19 when the virus attacks. However, it requires sophisticated biological design, trained personnel, bulky instruments, and is impossible or inaccurate to detect infected but recovered people or asymptomatic carriers. Here, in an effort towards expanding the screening coverage, we combined the extremely sensitive graphene field-effect transistor (Gr-FET) with highly selective antibody-antigen interaction to develop a COVID-19 immuno-sensor. The Gr-FET immunosensors can fast identify and accurately capture the subunit S1 protein from COVID-19 at a limit of detection down to 0.2 pM. Further results suggest that antigen S1 functionalized Gr-FET can be applied to screen neutralizing-antibodies with high efficiency. Thus, Gr-FET immunosensors provide an appealing opportunity to address the earlier diagnosis as well as the analysis and design of neutralizing-antibody locking methods of this ongoing public health crisis.

Label-Free Fluorescence Molecular Beacon Probes Based on G-Triplex DNA and Thioflavin T for Protein Detection.

Protein detection plays an important role in biological and biomedical sciences. The immunoassay based on fluorescence labeling has good specificity but a high labeling cost. Herein, on the basis of G-triplex molecular beacon (G3MB) and thioflavin T (ThT), we developed a simple and label-free biosensor for protein detection. The biotin and streptavidin were used as model enzymes. In the presence of target streptavidin (SA), the streptavidin hybridized with G3MB-b (biotin-linked-G-triplex molecular beacon) perfectly and formed larger steric hindrance, which hindered the hydrolysis of probes by exonuclease III (Exo III). In the absence of target streptavidin, the exonuclease III successively cleaved the stem of G3MB-b and released the G-rich sequences which self-assembled into a G-triplex and subsequently activated the fluorescence signal of thioflavin T. Compared with the traditional G-quadruplex molecular beacon (G4MB), the G3MB only needed a lower dosage of exonuclease III and a shorter reaction time to reach the optimal detection performance, because the concise sequence of G-triplex was good for the molecular beacon design. Moreover, fluorescence experiment results exhibited that the G3MB-b had good sensitivity and specificity for streptavidin detection. The developed label-free biosensor provides a valuable and general platform for protein detection.

Development of Gold Nanoparticle Micropatterns for the Electrical Detection of Proteins.

Protein analysis can be used to efficiently detect the early stages of various diseases. However, conventional protein detection platforms require expensive or complex equipment, which has been a major obstacle to their widespread application. In addition, uncertain signals from non-specific adhesion interfere with the precise interpretation of the results. To overcome these problems, the development of a technique that can detect the proteins in a simple method is needed. In this study, a platform composed of gold nanoparticles (GNPs) was fabricated through a simple imprinting method for protein detection. The corrugated surface naturally formed by the nanoparticle assemblies simultaneously increases the efficiency of adhesion and binding with analytes and reduces undesired interactions. After forming the GNP micropatterns, post-functionalization with both cationic and neutral ligands was performed on the surface to manipulate their electrostatic interaction with proteins. Upon protein binding, the change in the electrical values of the micropatterns was recorded by using a resistance meter. The resistance of the positively charged micropatterns was found to increase due to the electrostatic interaction with proteins, while no significant change in resistance was observed for the neutral micropatterns after immersion in a protein solution. Additionally, the selective adsorption of fluorescent proteins onto the micropatterns was captured using confocal microscopy. These simply imprinted GNP micropatterns are sensitive platforms that can detect various analytes by measuring the electrical resistance with portable equipment.

A low-cost electrochemical biosensor platform for C-reactive protein detection

Antibiotic resistance is a worldwide problem aggravated by the overuse of prophylactic antibiotic therapies and lack of real-time biosensing equipment to differentiate viral from bacterial infections at the Point of Care (POC), particularly in rural areas with limited access to clinical laboratory facilities. As recent studies reveal the potential of C-Reactive Protein (CRP) and other acute phase biomarkers to achieve early determination of etiology in acute febrile illness, novel biosensing equipment become a plausible approach in preventing the unnecessary prescription of antibiotics. A low-cost experimental platform was engineered to measure CRP concentrations in 50 μL samples of buffer and enriched plasma, using label-free antigenic probes, legacy electrochemical methods and open-source hardware. The prototype presents a portable, cost-effective device for use at the POC with a simplified user interface that can be used wirelessly by healthcare professionals using a mobile phone or laptop. Based on the measured CRP levels, the device suggests if the acute febrile episode is likely to be of bacterial etiology.

Gold Nanoparticles/Thermochromic Composite Film on Screen-Printed Electrodes for Simultaneous Detection of Protein and Temperature

In this study, gold nanoparticles and thermochromic composite films modified screen-printed carbon electrodes (TM-AuNPsSPCEs) were developed as a platform for the simultaneous detection of protein and temperature. The TM-AuNPs composited film had better sensitivity resulting from the gold nanoparticles amplification effect. A phase transition model analysis of TM-AuNPs films found that the TM-AuNPs films had three-phase transition intervals (80℃) which accommodated the temperature requirements for protein denaturation. When used to detect different concentrations of haemoglobin (Hb) solution, the TM-AuNPs modified SPCEs had a better sensitivity in detecting the different concentrations in comparison to TM and AuNP modified SPCEs which showed no clear sensitivity towards the different Hb concentrations. The dual detection and excellent sensitivity show a good application prospect for the study of the TM-AuNPs composite film.

Graphene oxide/perylene-aniline electrochemiluminescence platform for protein detection based on molecule recognition.

Most biosensors for folate receptor (FR) detection based on folic acid (FA) recognition usually contain FA-linked single-strand DNA (FA-ssDNA) and nuclease to promote sensitivity, which increases expenses and involves complicated assay processes. A few electrochemiluminescence (ECL) sensors that do not use FA-ssDNA and nuclease directly graft FA onto an ECL nanomaterial through covalent bonding for FR detection. In this study, we used FA-ssDNA to non-covalently graft FA onto π-conjugated ECL nanomaterial graphene oxide (GO)/perylene-aniline for fabricating ultrasensitive FR sensors without nuclease. 3,4,9,10-Perylenetetracarboxylic dianhydride (PTCDA) and aniline (An) self-assembled into π-conjugated nanorods, which were then loaded onto GO. This material was reported to produce 673 nm-dominated ECL with the co-reactant K2S2O8, and was used as an ECL platform. FA-modified Poly-dA-ssDNA (FA-Poly-dA-ssDNA) molecules, consisting of 20 bases, were attached to the surface of GO/PTCDA-An to capture FR. A significant decrease of ECL intensity was observed due to the steric hindrance of FR. The proposed sensors exhibited high detection sensitivity with a linear range from 1 fg mL-1 to 1 ng mL-1 and a detection limit of 0.636 fg mL-1. The sensors also showed good potential in real sample detection. Without introducing nuclease and complicated chemical reactions, this work provides a new sensing strategy for protein detection based on molecular recognition, which is extremely important in clinical diagnosis.

Ratiometric Detection of DNA and Protein in Serum by a Universal Tripyridinyl RuII Complex-Encapsulated SiO2@Polydopamine Fluorescence Nanoplatform.

Fluorescence ratiometric biosensors are valuable tools for the accurate and sensitive prediction and diagnosis of diseases. However, seldom have fluorescence ratiometric biosensors for protein and DNA been reported because of the shortage of suitable nanoscale scaffolds. Herein, a tripyridinyl RuII complex-encapsulated SiO2@polydopamine (Ru-SiO2@PDA) nanocomposite was designed as a universal platform for fluorescence ratiometric detection of DNA and protein in serum samples. The Ru-SiO2@PDA nanocomposites have a narrow size distribution, exhibit good biosafety, and are convenient for the postmodification of biorecognition elements. Under irradiation, they can emit a stable and strong luminescence at 650 nm and simultaneously quench the fluorescence emitted from the fluorophores getting close to them. Once the capture probes such as single-stranded DNA and aptamer are assembled, the fluorophores labeled on them are then brought close to their PDA shell and quenched. However, the biorecognition behaviors change the probe's configuration and take the fluorophore far away from the PDA shell. Correspondingly, the fluorescence recovers and its ratio to the constant fluorescence reference is linear to the targets' concentration. Using a D-catalyst and thrombin as model analytes, the Ru-SiO2@PDA-based nanoplatform shows high sensitivity and good accuracy in the serum sample analysis. Regarding these attractive properties, the Ru-SiO2@PDA nanoplatform provides a new avenue for the accurate and sensitive fluorescence assay of a wide range of targets in complex systems.

Abstract PO-045: Multiplex analysis of coronavirus and influenza antibodies using the ZIVA multiplex platform

Abstract Detection of antibodies to upper respiratory pathogens is critical to surveillance, assessment of the immune status of individuals, vaccine development, and basic biology. The urgent need for antibody detection tools has proven particularly acute in the COVID-19 era. We have used Arrayed Imaging Reflectometry (AIR™), a label-free protein microarray technology that relies on the target binding-induced perturbation of an antireflective coating on the surface of a silicon chip, to create a multiplex antibody detection array. ZIVA is a new multiplex protein detection platform that leverages AIR™ technology. We have used ZIVA technology to develop an acute respiratory virus panel capable of detecting the presence of antibodies specific for coronavirus and influenza including, but not limited to, detection of antibodies to SARS-CoV-2, SARS-CoV-1, MERS, three circulating coronavirus strains (HKU1, 229E, OC43), and multiple strains of influenza. We find that the array is readily able to distinguish uninfected from convalescent COVID-19 subjects and provides quantitative information about total IgG, as well as IgG- and IgM-specific responses. Samples may also be evaluated with ZIVA cytokine assays to monitor the cytokine storm in conjunction with viral antibody response. Citation Format: Jon Schmuke, Ben Miller, Linda Meeh, Byran Witherbee. Multiplex analysis of coronavirus and influenza antibodies using the ZIVA multiplex platform [abstract]. In: Proceedings of the AACR Virtual Meeting: COVID-19 and Cancer; 2020 Jul 20-22. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(18_Suppl):Abstract nr PO-045.

Abstract 1127: Adarza's ZIVA multiplex protein detection platform utilizes a label-free technology to simultaneously monitor murine and human inflammation cytokines in a CAR-T model system

Abstract We have used Arrayed Imaging Reflectometry (AIR™), a label-free protein microarray technology that relies on the target binding-induced perturbation of an antireflective coating on the surface of a silicon biosensor, to create a multiplex antibody array for the detection of human and murine serum cytokines and inflammatory markers. The biosensor is capable of providing quantitative protein concentrations, with limits of detection in the low picograms per mL. Provided in a standard 96-well microplate format, the label-free nature of the array enables flexible creation of standard or custom panels for development of model systems. Here, we demonstrate a custom 22 plex panel developed in collaboration with WUGEN Inc. to provide multi-species cytokine analysis in humanized CAR-T cell models for the evaluation of biologic product efficacy and safety. Samples from in vitro and in vivo models developed for the evaluation of CAR-T activity were analyzed and demonstrated a novel platform for concurrent cross-species cytokine analysis on a single array. Results are evaluated for sensitivity, precision, and accuracy with ZIVA assays comparable to traditional ELISAs. With array capabilities from low to hundreds of plex combined with a hands-free, automation-friendly workflow enabled by the ZIVA instrument, the technology has been designed for the ultimate user-experience. Citation Format: Jon J. Schmuke, Alun Carter, You Zhou, Ernest Mueller, Matt Cooper. Adarza's ZIVA multiplex protein detection platform utilizes a label-free technology to simultaneously monitor murine and human inflammation cytokines in a CAR-T model system [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 1127.

Adarza’s ZIVA multiplex protein detection platform demonstrates flexibility with custom targets for monitoring both inflammation and anti-drug antibody response

Abstract We have used Arrayed Imaging Reflectometry (AIR™), a label-free protein microarray technology that relies on the target binding-induced perturbation of an antireflective coating on the surface of a silicon chip, to create a multiplex antibody array. ZIVA is a new multiplex protein detection platform that leverages AIR™ technology. Understanding the immunogenicity of a therapeutic protein is critical to the development of bioactive stability and efficacy. ZIVA technology can be customized to monitor for multiple biomarker types including markers of inflammation as well as specific ADA targets. In this example, biosensors were custom printed to monitor for the presence of an anti-drug antibodies generated against a single chain antibody presented on the surface of engineered CAR-T cells. Samples that are positive for ADA response may also be evaluated for presence of neutralizing antibodies (NAB) by evaluating specific controls also included on the biosensors. With array capabilities from low to hundreds of plex combined with a hands-free, automation-friendly workflow enabled by the ZIVA instrument, the technology has been designed for the ultimate user-experience.

Adarza’s ZIVA multiplex protein detection platform offers format for ultra-high sensitivity

Abstract We have used Arrayed Imaging Reflectometry (AIR™), a label-free protein microarray technology that relies on the target binding-induced perturbation of an antireflective coating on the surface of a silicon chip, to create a multiplex antibody array for the detection of both low and highly abundant targets. ZIVA is a new multiplex protein detection platform that leverages AIR™ technology. Because of the label-free properties, sensitivity can be “fine-tuned” by leveraging a variety of mass enhancing options. We have previously demonstrated assays that respond in the nanogram to picogram/ml sensitivity using one format for enhancement. Herein, we demonstrate a newly developed mass enhancement option that increases sensitivity from very low pg/ml to mid-femtogram/ml levels. A custom designed biosensor with markers for inflammation and other custom needs was used to demonstrate this ultra-high sensitivity format in a multiplex array. With array capabilities from low to hundreds of plex combined with a hands-free, automation-friendly workflow enabled by the ZIVA instrument, the technology has been designed for the ultimate user-experience.

Adarza’s ZIVA multiplex protein detection platform utilizes a label-free technology to simultaneously monitor murine and human inflammation cytokines in a CAR-T model system

Abstract We have used Arrayed Imaging Reflectometry (AIR™), a label-free protein microarray technology that relies on the target binding-induced perturbation of an antireflective coating on the surface of a silicon biosensor, to create a multiplex antibody array for the detection of human and murine serum cytokines and inflammatory markers. The biosensor is capable of providing quantitative protein concentrations, with limits of detection in the low picograms per mL. The flexibility of this system can also reduce the need to generate a series of sample dilutions in order to get results in a limited detection range by enabling the development of each assay with sensitivity expectations for each target on a chip. Provided in a standard 96-well microplate format, the label-free nature of the array enables flexible creation of standard or custom panels for development of model systems. Here, we demonstrate a custom 22 plex panel developed in collaboration with WUGEN Inc. to provide multi-species cytokine analysis in humanized CAR-T cell models for the evaluation of biologic product efficacy and safety. Samples from in vitro and in vivo models developed for the evaluation of CAR-T activity were analyzed and demonstrated a novel platform for concurrent cross-species cytokine analysis on a single array. Results are evaluated for sensitivity, precision, and accuracy with ZIVA assays comparable to traditional ELISAs. With array capabilities from low to hundreds of plex combined with a hands-free, automation-friendly workflow enabled by the ZIVA instrument, the technology has been designed for the ultimate user-experience.

A disposable microfluidic-integrated hand-held plasmonic platform for protein detection

Healthcare is in the midst of a transformative shift from centralized care to point-of-care (POC). In this regard, recent efforts have focused on integration of biosensing technologies with clinical management and existing healthcare systems to improve the effectiveness and quality of care. Plasmonic technologies in particular, have been used for multiple applications in biosensing, pharmaceutical industry, food quality monitoring, and healthcare. However, bulky-sized platforms, expensive instrumentation, incomprehensive benchmarking, laborious protocols, and time-consuming processing steps remain challenges to adopt biosensing platforms to the POC settings. Here, we present a hand-held biosensing platform that integrates a plasmonic detection modality with a microfluidic chip. As a biological target model, we assess hemoglobin—an iron carrying protein in red blood cells. We comprehensively perform theoretical simulations and kinetic calculations to benchmark the platform performance. Overall, this miniaturized platform provides label-free detection, simple configuration for user-interface, facile sampling, assay-time down to 15–30 min, and inexpensive disposable chips. Therefore, this platform will potentially accelerate the deployment of portable biosensing systems for the POC and primary care settings.

Flow-Cytometry Platform for Intracellular Detection of FVIII in Blood Cells: A New Tool to Assess Gene Therapy Efficiency for Hemophilia A

Detection of factor VIII (FVIII) in cells by flow cytometry is controversial, and no monoclonal fluorescent antibody is commercially available. In this study, we optimized such an assay and successfully used it as a platform to study the functional properties of phosphoglycerate kinase (PGK)-FVIII lentiviral vector-transduced cells by directly visualizing FVIII in cells after different gene transfer conditions. We could measure cellular stress parameters after transduction by correlating gene expression and protein accumulation data. Flow cytometry performed on transduced cell lines showed that increasing MOI rates resulted in increased protein levels, plateauing after an MOI of 30. We speculated that, at higher MOI, FVIII production could be impaired by a limiting factor required for proper folding. To test this hypothesis, we interfered with the unfolded protein response by blocking proteasomal degradation and measured the accumulation of intracellular misfolded protein. Interestingly, at higher MOIs the cells displayed signs of toxicity with reactive oxygen species accumulation. This suggests the need for identifying a safe window of transduction dose to avoid consequent cell toxicity. Herein, we show that our flow cytometry platform for intracytoplasmic FVIII protein detection is a reliable method for optimizing gene therapy protocols in hemophilia A by shedding light on the functional status of cells after gene transfer.

Robust and Universal SERS Sensing Platform for Multiplexed Detection of Alzheimer's Disease Core Biomarkers Using PAapt-AuNPs Conjugates.

Multiplexed detection of Alzheimer's disease (AD) core biomarkers is of great significance to early diagnosis and personalized treatment of AD patients. Herein, we construct a robust and convenient surface-enhanced Raman scattering (SERS) biosensing platform for simultaneous detection of Aβ(1-42) oligomers and Tau protein using different Raman dye-coded polyA aptamer-AuNPs (PAapt-AuNPs) conjugates. This strategy relies on the specific protein-aptamer binding-mediated aggregation of AuNPs and the concomitant plasmonic coupling effect that allow us to "turn on" SERS detection of protein biomarkers. To the best of our knowledge, this is the first work in which PAapt-AuNPs conjugates are used for probing protein biomarkers, which may be enlightening for the exploitation of more extensive biological applications of aptamer-AuNPs conjugates. The results reveal that the present strategy displays excellent analytical performance. Moreover, the applicability of this strategy is demonstrated in the artificial cerebrospinal fluid (CSF) samples with satisfactory results. Except for the prominent sensitivity and practicality, our strategy offers additional advantages. The preparation of nanoconjugates is handy and easily repeated, and the synthesis cost is greatly reduced because it dispenses with the complicated labeling process. Moreover, the assay can be accomplished in 15 min, allowing rapid detection of protein biomarkers. Furthermore, simultaneous detection of Tau protein and Aβ(1-42) oligomers is realized by employing different Raman dye-coded nanoconjugates, which is valuable for accurately predicting and diagnosing AD disease. Thus, our PAapt-AuNPs conjugate-based multiplexed SERS strategy indeed creates a useful and universal platform for detecting multiple protein biomarkers and related clinical diagnosis.

C-reactive protein detection based on ISFET structure with gate dielectric SiO2-CeO2

This paper considers a new biosensor based on p-channel ion sensitive field effect transistors (ISFET) as a sensitive platform for low concentration detection of C-reactive protein (CRP). The ISFET structure is fabricated by a standard CMOS technology on a phosphorus-doped Si (111) wafer with ρ = 4.5 Ω.cm. A complex layer of gate dielectric SiO2-CeO2 (dSiO2 = 50 nm, dCeO2 = 10 nm) is chosen since such layers have previously proven their suitability and are technologically simple to manufacture. To improve the antigen binding to the sensitive surface, streptavidin and biotin with and without melanin agents are immobilized on the gate dielectric. Optical microscopy, SEM and AFM results showed that the immobilized streptavidin and biotin agents form porous dendrite structure. The limit of CRP detection is 0.1 mg/L, the range of measuring CRP concentrations is 0.1–2.5 mg/L, the measurement time is about 1 min, and the required amount of solution for analysis - 20 μL. The application of the sensor was evaluated using real samples of human blood serum.

Proximity ligation assays for precise quantification of femtomolar proteins in single cells using self-priming microfluidic dPCR chip

The quantification of low concentration proteins can facilitate the discovery of some significant biomarkers, and provide us a more profound understanding of cell heterogeneity when applied to single cell analysis. However, most state-of- art single cell protein detection platforms are bulky, expensive and complicated. Here we report a simple and low cost microfluidic dPCR (digital polymerase chain reaction) chip-based proximity ligation assay (PLA) for the quantification of low concentration proteins. First, standard hCSTB (human cystatin B) protein was used to optimize the related experimental conditions. Comparing to ordinary PLA tests, the results showed that our method achieved femtomolar limit of detection (LOD) with a linear dynamic range over three to four orders of magnitude. Then human CD147 protein, a reported biomarker for hepatoma carcinoma, was detected in single HepG2 and L02 cells. The results showed that there were wide disparities in single cell CD147 abundance for both of the two cell lines. And the average CD147 protein content in single HepG2 cells displayed 2-fold increase in comparison to that in single L02 cells. Comparing to the research findings obtained at bulk level, our method can provide more useful information for diagnosis and targeted therapy of tumors.

(Invited) Detection of Heart Failure-Related Biomarker in Whole Blood with Graphene Field Effect Transistor Biosensor

Since brain natriuretic peptide (BNP) has become internationally recognized biomarkers in the diagnosis and prognosis of heart failure (HF), it is highly desirable to search for a novel sensing tool for detecting the patient’s BNP level at the early stage. Here we report a platinum nanoparticles (PtNPs)-decorated reduced graphene oxide (rGO) field effect transistor (FET) biosensor coupled with a microfilter system for label-free and highly sensitive detection of BNP in whole blood. The PtNPs-decorated rGO FET sensor was obtained by drop-casting rGO onto the pre-fabricated FET chip and subsequently assembling PtNPs on the graphene surface. After anti-BNP was bound to the PtNPs surface, BNP was successfully detected by the anti-BNP immobilized FET biosensor. It was found that the developed FET biosensor was able to achieve a low detection limitation of 100 fM. Moreover, BNP was successfully detected in human whole blood sample treated by a custom-made microfilter, suggesting the sensor’s capability of working in a complex sample matrix. The developed FET biosensor provides a new sensing platform for protein detection, showing its potential applications in clinic sample.

Detection of Single Proteins with a General Nanopore Sensor.

Single protein sensing based on solid-state nanopores is promising but challenging, because the fast translocation velocity of a protein is beyond the bandwidth of nanopore instruments. To decelerate the translocation speed, here, we employed a common protein cross-link interaction to achieve a general and robust nanopore sensing platform for single-molecule detection of protein. Benefiting from the EDC/NHS coupling interaction between nanopore and proteins, a 10-fold decrease in speed has been achieved. The clearly distinguishable current signatures further reveal that the anisotropictranslocationof a protein, which are horizontal, vertical, and flipping transit inside nanopore confinement. This strategy provides a general platform for rapid detection of proteins as well as exploring fundamental protein dynamics at the single-molecule level.