Protein-Induced DNA Dumbbell Amplification (PINDA) and its applications to food hazards detection.

Quantification of trace amounts of proteins is technically challenging because proteins cannot be directly amplified like nucleic acids. To improve the analytical sensitivity and to complement conventional protein analysis methods, we developed a highly sensitive and homogeneous detection strategy called Protein-Induced DNA Dumbbell Amplification (PINDA). PINDA combines protein recognition with exponential nucleic acid amplification by using protein binding probes made of DNA strands conjugated to protein affinity ligands. When a pair of probes bind to the same target protein, complementary nucleic acid sequences that are conjugated to each probe are brought into close proximity. The increased local concentration of the probes results in the formation of a stable dumbbell structure of the nucleic acids. The DNA dumbbell is readily amplifiable exponentially using techniques such as loop-mediated isothermal amplification. The PINDA assay eliminates the need for washing or separation steps, and is suitable for on-site applications. Detection of the model protein, thrombin, has a linear range of 10 fM-100 pM and detection limit of 10 fM. The PINDA technique is successfully applied to the analysis of dairy samples for the detection of ß-lactoglobulin, a common food allergen, and Salmonella enteritidis, a foodborne pathogenic bacterium. The PINDA assay can be easily modified to detect other targets by changing the affinity ligands used to bind to the specific targets.

A quantitative method for aquaporin-1 protein using magnetic preconcentration and probe-based immunoassay coupling to inductively coupled plasma mass spectrometry in urine analysis.

BACKGROUND: Aquaporin-1 (AQP1) protein plays a crucial role in intracellular and extracellular water homeostasis and fluid transport in organs and tissues associated with diverse life activities and is extremely abundant in the kidney. Accurate detection of AQP1 in urine can be applied as screening of early-stage disease. Application of magnetic preconcentration and probe-based signal amplification strategy coupling to inductively coupled plasma mass spectrometry (ICP-MS) is a more accurate, sensitive and specific detection method for AQP1 in complex biological samples compared to conventional methods. RESULTS: We described an element-labelling strategy based on magnetic preconcentration and probe-based immunoassay coupling to ICP-MS detection. The magnetic beads (MBs) modified with epoxy groups were capable of enriching AQP1 proteins and separating them from complex matrices. The probe constructed by conjugating anti-AQP1 antibody molecules on the surface of gold nanoparticles could specifically recognize AQP1 proteins attached on MBs and be analyzed by ICP-MS. The concentration of AQP1 protein could be precisely quantified and amplified by 14,000 times through the corresponding signal of Au atoms. This assay for AQP1 protein quantification achieved a detection limit down to 0.023 ng mL-1, a broad linear calibration curve between 0.3 ng mL-1 and 30 ng mL-1, as well as outstanding specificity. SIGNIFICANCE: The proposed method was successfully applied to detect AQP1 protein in human urine samples, showing the potential for its applications concerning accurate AQP1 quantification. It can also screen a wide range of proteins provided the antibodies specific to these target proteins are available.

AFM-fishing technology for protein detection in solutions.

The review considers the possibility of using atomic force microscopy (AFM) as a basic method for protein detection in solutions with low protein concentrations. The demand for new bioanalytical approaches is determined by the problem of insufficient sensitivity of systems used in routine practice for protein detection. Special attention is paid to demonstration of the use in bioanalysis of a combination of AFM and fishing methods as an approach of concentrating biomolecules from a large volume of the analyzed solution on a small surface area.

Label-free and label-based electrochemical detection of disease biomarker proteins.

Introduction: Biosensors, analytical devices integrating biological sensing elements with physicochemical transducers, have gained prominence as rapid and convenient tools for monitoring human health status using biochemical analytes. Due to its cost-effectiveness, simplicity, portability, and user-friendliness, electrochemical detection has emerged as a widely adopted method in biosensor applications. Crucially, biosensors enable early disease diagnosis by detecting protein biomarkers associated with various conditions. These biomarkers offer an objective indication of medical conditions that can be accurately observed from outside the patient. Method: This review comprehensively documents both label-free and labelled detection methods in electrochemical biosensor techniques. Label-free detection mechanisms elicit response signals upon analyte molecule binding to the sensor surface, while labelled detection employs molecular labels such as enzymes, nanoparticles, and fluorescent tags. Conclusion: The selection between label-free and labelled detection methods depends on various factors, including the biomolecular compound used, analyte type and biological binding site, biosensor design, sample volume, operational costs, analysis time, and desired detection limit. Focusing on the past six years, this review highlights the application of label-free and labelled electrochemical biosensors for detecting protein biomarkers of diseases.

Review of single-molecule immunoassays: Non-chip and on-chip Assays.

Enhancing the sensitivity of immunoassays is an important requirement in the field of immunology, especially in light of rapid developments in genetic testing, making the detection of low-abundance protein biomarkers crucial. Therefore, innovations in highly sensitive immunoassays are imperative. This demand has led to the emergence of single-molecule immunoassays (SMIs), driving advancements in early diagnostic techniques, and ushering in a new era of immunoassays. This review begins by tracing the development of immunoassays and offers a detailed discussion of SMI technology across two distinct pathways: non-chip (SMI without microfluidic chips) and on-chip (SMI with microfluidic chips). Furthermore, we evaluated and compared these methods using two pathways. In addition, this review discusses the significance of SMI techniques in the diagnosis of various diseases and their current applications in laboratory and clinical settings. The progress of SMI in commercial applications and suggestions for innovative directions are also summarized. Despite the considerable potential of SMI, these technologies face challenges in practical application, particularly in developing countries and economically disadvantaged regions. The final section of this review addresses the challenges and prospects of these technologies.

Imaging Diffractometric Biosensors for Label-Free, Multi-Molecular Interaction Analysis.

Biosensors are used for the specific and sensitive detection of biomolecules. In conventional approaches, the suspected target molecules are bound to selected capture molecules and successful binding is indicated by additional labelling to enable optical readout. This labelling requires additional processing steps tailored to the application. While numerous label-free interaction assays exist, they often compromise on detection characteristics. In this context, we introduce a novel diffractometric biosensor, comprising a diffractive biosensor chip and an associated optical reader assembly. This innovative system can capture an entire assay, detecting various types of molecules in a label-free manner and present the results within in a single, comprehensive image. The applicability of the biosensor is assessed for the detection of viral DNA as well as proteins directly in human plasma, investigating different antigens. In our experiments, we achieve a detection limit of 4.2 pg/mm², which is comparable to other label-free optical biosensors. The simplicity and robustness of the method make it a compelling option for advancing biosensing technologies. This work contributes to the development of an imaging diffractometric biosensor with the potential for multiple applications in molecular interaction analysis.

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.

One-Pot Detection of Proteins Using a Two-Way Extension-Based Assay with Cas12a.

Protein biomarkers are an important class of biomarkers in disease diagnosis and are traditionally detected by enzyme-linked immunosorbent assay and mass spectrometry, which involve multiple steps and a complex workflow. In recent years, many CRISPR-Cas12a-based methods for protein detection have been developed; however, most of them have not overcome the workflow complications observed in traditional assays, limiting their applicability in point-of-care testing. In this work, we designed a single-step, one-pot, and proximity-based isothermal immunoassay integrating CRISPR Cas12a for homogeneous protein target detection with a simplified workflow and high sensitivity. Probes consisting of different binders (small molecule, aptamer, and antibody) conjugated with oligonucleotides undergo two-way extension upon binding to the protein targets, leading to downstream DNA amplification by a pair of nicking enzymes and polymerases to generate target sequences for Cas12a signal generation. We used the streptavidin-biotin model to demonstrate the design of our assay and proved that all three elements of protein detection (target protein binding, DNA amplification, and Cas12a signal generation) could coexist in one pot and proceed isothermally in a single buffer system at a low reaction volume of 10 µL. The plug-and-play applicability of our assay has been successfully demonstrated using four different protein targets, streptavidin, PDGF-BB, antidigoxigenin antibody, and IFNγ, with the limit of detection ranging from fM to pM.

Highly sensitive and label-free protein immunoassay-based biosensor comprising infrared metamaterial absorber inducing strong coupling.

A mid-infrared label-free immunoassay-based biosensor is an effective device to help identify and quantify biomolecules. This biosensor employs a surface-enhanced infrared absorption spectroscopy, which is a highly potent sensing technique for detecting minute quantities of analytes. In this study, a biosensor was constructed using a metamaterial absorber, which facilitated strong coupling effects. For maximum coupling effect, it is necessary to enhance the near-field intensity and the spatial and spectral overlap between the optical cavity resonance and the vibrational mode of the analyte. Due to significant peak splitting, conventional baseline correction methods fail to adequately analyze such a coupling system. Therefore, we employed a coupled harmonic oscillation model to analyze the spectral distortion resulting from the peak splitting induced by the strong coupling effect. The proposed biosensor with a thrombin-binding aptamer-based immunoassay could achieve a limit of detection of 267.4 pM, paving the way for more efficient protein detection in clinical practice.

Development of a split-luciferase assay to establish optimal protein secretion conditions for protein production by Bacillus subtilis.

Bacillus subtilis is a Gram-positive bacterium that is frequently used in the bioindustry for the production of various proteins, because of its superior protein secretion capacities. To determine optimal conditions for protein secretion by B. subtilis, a quick and sensitive method for measuring protein secretion is crucial. A fast and universal assay is most useful for detecting diverse proteins in a high-throughput manner. In this study, we introduce a split-luciferase-based method for measuring protein secretion by B. subtilis. The NanoBiT system was used to monitor secretion of four different proteins: xylanase A, amylase M, protein glutaminase A, and GFP nanobody. Our findings underscore the split-luciferase system as a quick, sensitive, and user-friendly method.

Enhancing the understanding of SARS-CoV-2 protein with structure and detection methods: An integrative review.

As the rapid and accurate screening of infectious diseases can provide meaningful information for outbreak prevention and control, as well as owing to the existing limitations of the polymerase chain reaction (PCR), it is imperative to have new and validated detection techniques for SARS-CoV-2. Therefore, the rationale for outlining the techniques used to detect SARS-CoV-2 proteins and performing a comprehensive comparison to serve as a practical benchmark for future identification of similar viral proteins is clear. This review highlights the urgent need to strengthen pandemic preparedness by emphasizing the importance of integrated measures. These include improved tools for pathogen characterization, optimized societal precautions, the establishment of early warning systems, and the deployment of highly sensitive diagnostics for effective surveillance, triage, and resource management. Additionally, with an improved understanding of the virus' protein structure, considerable advances in targeted detection, treatment, and prevention strategies are expected to greatly improve our ability to respond to future outbreaks.

Antibody Design for the Quantification of Photosynthetic Proteins and Their Isoforms.

Antibodies are a valuable research tool, with uses including detection and quantification of specific proteins. By using peptide fragments to raise antibodies, they can be designed to differentiate between structurally similar proteins, or to bind conserved motifs in divergent proteins. Peptide sequence selection and antibody validation are crucial to ensure reliable results from antibody-based experiments. This chapter describes the steps for the identification of peptide sequences to produce protein- or isoform-specific antibodies using recombinant technologies as well as the subsequent validation of such antibodies. The photosynthetic protein Rubisco activase is used as a case study to explain the various steps involved and key aspects to take into consideration.

A Study of the Drift Phenomena of Gate-Functionalized Biosensors and Dual-Gate-Functionalized Biosensors in Human Serum.

In this paper, we study the drift behavior of organic electrochemical transistor (OECT) biosensors in a phosphate-buffered saline (PBS) buffer solution and human serum. Theoretical and experimental methods are illustrated in this paper to understand the origin of the drift phenomenon and the mechanism of ion diffusion in the sensing layer. The drift phenomenon is explained using a first-order kinetic model of ion adsorption into the gate material and shows very good agreement with experimental data on drift in OECTs. We show that the temporal current drift can be largely mitigated using a dual-gate OECT architecture and that dual-gate-based biosensors can increase the accuracy and sensitivity of immuno-biosensors compared to a standard single-gate design. Specific binding can be detected at a relatively low limit of detection, even in human serum.

Highly Multiplexing, Throughput and Efficient Single-Cell Protein Analysis with Digital Microfluidics.

Proteins as crucial components of cells are responsible for the majority of cellular processes. Sensitive and efficient protein detection enables a more accurate and comprehensive investigation of cellular phenotypes and life activities. Here, a protein sequencing method with high multiplexing, high throughput, high cell utilization, and integration based on digital microfluidics (DMF-Protein-seq) is proposed, which transforms protein information into DNA sequencing readout via DNA-tagged antibodies and labels single cells with unique cell barcodes. In a 184-electrode DMF-Protein-seq system, ≈1800 cells are simultaneously detected per experimental run. The digital microfluidics device harnessing low-adsorbed hydrophobic surface and contaminants-isolated reaction space supports high cell utilization (>90%) and high mapping reads (>90%) with the input cells ranging from 140 to 2000. This system leverages split&pool strategy on the DMF chip for the first time to overcome DMF platform restriction in cell analysis throughput and replace the traditionally tedious bench-top combinatorial barcoding. With the benefits of high efficiency and sensitivity in protein analysis, the system offers great potential for cell classification and drug monitoring based on protein expression at the single-cell level.

Utilizing Magnetic Levitation to Detect Lung Cancer-Associated Exosomes.

Extracellular vesicles, especially exosomes, have attracted attention in the last few decades as novel cancer biomarkers. Exosomal membrane proteins provide easy-to-reach targets and can be utilized as information sources of their parent cells. In this study, a MagLev-based, highly sensitive, and versatile biosensor platform for detecting minor differences in the density of suspended objects is proposed for exosome detection. The developed platform utilizes antibody-functionalized microspheres to capture exosomal membrane proteins (ExoMPs) EpCAM, CD81, and CD151 as markers for cancerous exosomes, exosomes, and non-small cell lung cancer (NSCLC)-derived exosomes, respectively. Initially, the platform was utilized for protein detection and quantification by targeting solubilized ExoMPs, and a dynamic range of 1-100 nM, with LoD values of 1.324, 0.638, and 0.722 nM for EpCAM, CD81, and CD151, were observed, respectively. Then, the sensor platform was tested using exosome isolates derived from NSCLC cell line A549 and MRC5 healthy lung fibroblast cell line. It was shown that the sensor platform is able to detect and differentiate exosomal biomarkers derived from cancerous and non-cancerous cell lines. Overall, this innovative, simple, and rapid method shows great potential for the early diagnosis of lung cancer through exosomal biomarker detection.

Western blotting: evolution of an old analytical method to a new quantitative tool for biomarker measurements.

Western blotting (WB) is a widely used laboratory technique for detecting specific proteins in biological matrices. Recent advances in antibody production, automation, gel and membrane manufacturing and highly sensitive detection platforms have transformed WB from a labor-intensive and qualitative method into a highly reproducible and quantitative assay suitable for biomarker detection. Despite these significant improvements in the capabilities and efficiency of WB, there remain challenges that hinder its widespread application as a research, diagnostic (in two-tiered assays like Lyme disease testing) and drug development tool. This article describes recent innovations introduced to WB methodology and the remaining challenges that prevent its wider adoption for biomarker measurements throughout the drug development process.

Comparative analysis of aptamers binding to SARS-CoV-2 N protein using capillary electrophoresis and bio-layer interferometry.

Aptamers are increasingly employed in SARS-CoV-2 theragnostics in recent years. Characterization of aptamers, testing affinity and kinetic parameters (e.g., equilibrium dissociation constant (KD), kon, and koff), can be done by several methods and influenced by many factors. This study aims to characterize the binding of aptamers to SARS-CoV-2 nucleocapsid (N) protein using capillary electrophoresis (CE) and bio-layer interferometry (BLI). These two analytical methods differ by how the aptamer binds to its target protein once the aptamer, as a capture ligand, is partitioned in solution (CE) or immobilized on the biosensor (BLI). With CE, the KD values of the N-binding aptamers (tNSP1, tNSP2, and tNSP3) were determined to be 18 ± 4 nM, 45 ± 11 nM, and 32 ± 7 nM, respectively, while the KD measurements by BLI yielded 4.8 ± 0.6, 4.5 ± 0.5, and 2.9 ± 0.3 nM, respectively. CE results showed a higher KD across all aptamers tested. The differences in the steric hindrance and confirmational structures of the aptamers immobilized on the BLI biosensors versus those suspended in the CE sample solution affect the molecular interactions between aptamers and the target proteins. Moreover, the buffer composition including pH and ionic strength can influence the stability of aptamer structures, or aptamer-protein complexes. All these variables affect the binding and calculated KD. In this sense, a KD value alone is not sufficient to make comparisons between aptamers; instead, the entire experimental setup should also be considered. This is particularly important when implementing aptamers in different bioanalytical systems.

Real-time and quantitative protein detection via polyacrylamide gel electrophoresis and online intrinsic fluorescence imaging.

The detection of intrinsic protein fluorescence is a powerful tool for studying proteins in their native state. Thanks to its label-free and stain-free feature, intrinsic fluorescence detection has been introduced to polyacrylamide gel electrophoresis (PAGE), a fundamental and ubiquitous protein analysis technique, to avoid the tedious detection process. However, the reported methods of intrinsic fluorescence detection were incompatible with online PAGE detection or standard slab gel. Here, we fulfilled online intrinsic fluorescence imaging (IFI) of the standard slab gel to develop a PAGE-IFI method for real-time and quantitative protein detection. To do so, we comprehensively investigated the arrangement of the deep-UV light source to obtain a large imaging area compatible with the standard slab gel, and then designed a semi-open gel electrophoresis apparatus (GEA) to scaffold the gel for the online UV irradiation and IFI with low background noise. Thus, we achieved real-time monitoring of the protein migration, which enabled us to determine the optimal endpoint of PAGE run to improve the sensitivity of IFI. Moreover, online IFI circumvented the broadening of protein bands to enhance the separation resolution. Because of the low background noise and the optimized endpoint, we showcased the quantitative detection of bovine serum albumin (BSA) with a limit of detection (LOD) of 20 ng. The standard slab gel provided a high sample loading volume that allowed us to attain a wide linear range of 0.03-10 µg. These results indicate that the PAGE-IFI method can be a promising alternative to conventional PAGE and can be widely used in molecular biology labs.

CRISPR/Cas12a linked sandwich aptamer assay for sensitive detection of thrombin.

BACKGROUND: Thrombin is a serine protease and hemostasis regulator with multiple functions and recognized as an important biomarker for diseases, and sensitive detection of thrombin is of significance for clinical diagnostics and disease monitoring. Recently, the target-triggered nonspecific single-stranded deoxyribonuclease activity of CRISPR/Cas system is discovered, making it become a powerful tool in assay developments due to the ease of signal amplification. In the short period of development, many CRISPR based nucleic acid detection methods have already played a critical role in clinical diagnostics. However, the application of CRISPR/Cas system for protein biomarkers remains limited. RESULTS: Here we describe a CRISPR/Cas12a linked sandwich aptamer assay for detection of thrombin, which was based on the formation of a sandwich complex of target by using a capture aptamer or antibody coated on the microplate and a well-designed detection DNA strand. The detection DNA strand contained an anti-thrombin aptamer and an active DNA of Cas12a, thus the sandwich complex was labeled with the active DNA. The active DNA triggered activity of Cas12a in indiscriminately cleaving fluorophore and quencher labeled DNA reporters, causing significant fluorescence increase. Our method enabled sensitive detection of thrombin down to 10 pM, and it showed high selectivity for thrombin. The assay exhibited good performance in diluted serum samples, demonstrating the applicability for thrombin analysis in the real media. SIGNIFICANCE: This assay combines the merits of high affinity of aptamer, trans-cleavage activity of Cas12a, high selectivity of sandwich format analysis, and high-throughput detection of microplate assay, and it shows promise in applications.

Ratiometric electrochemical immunosensor for simultaneous detection of C-myc and Bcl-2 based on multi-role alloy composites.

A ratiometric electrochemical immunosensor is proposed for simultaneous detection of cellular-myelocytomatosis oncoprotein (C-myc) and B-cell lymphoma 2 (Bcl-2) via the potential-resolved strategy. It relied on multi-role co-loaded alloy composites (CLACs) and poly(3,4-ethylenedioxythiophene) (PEDOT)-graphene oxide (GO)-multiwalled carbon nanotubes (MWCNTs) (PGM) modified electrodes. CLACs with good catalytic and enzyme-like properties were synthesized in one step by loading tetramethylbenzidine (TMB) or methylene blue (MB) into Pt-Pd alloy and used as label materials. After immunological reactions, CLACs showed distinguishable dual differential pulse voltammetry signals at - 0.26 V and 0.38 V, corresponding to C-myc and Bcl-2, and the PGM had an electrochemical signal at 1.2 V, which could be used as a reference signal to construct a ratiometric sensor. CLACs had a satisfactory synergistic effect with the PGM, and eventually achieved quadruple signal amplification. Thus, benefiting from multiple magnification and ratiometric self-calibration functions, sensitive detections of C-myc and Bcl-2 were achieved, with detection limits as low as 0.5 and 2.5 pg mL-1, respectively. Additionally, when the designed method was applied to blood samples from lymphoma patients, results consistent with the ELISA kit were obtained. This will open avenues for constructing multiple protein detection sensors.